Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex

ABSTRACT

A method for identifying nucleic acid ligands to target molecules using the SELEX procedure. Nucleic acid candidate sequences contain photoreactive groups. After exposure of the nucleic acid sequences to the target molecule, nucleic acid-target molecule complexes are formed between nucleic acids having increased affinity to the target molecule and the target molecule. The complexes are irradiated such that photocrosslinks form between the photoreactive groups of the bound nucleic acids and the target molecule. The photocrosslinked complexes are separated from unbound nucleic acids, and the nucleic acids amplified to yield a ligand-enriched mixture of nucleic acids.  
     Described herein are methods for improved partitioning between high and low affinity nucleic acid ligands identified through the SELEX method, termed solution SELEX. The solution SELEX method achieves partitioning between high and low affinity nucleic acid-target complexes through a number of methods, including (1) primer extension inhibition which results in differentiable cDNA products. Primer extension inhibition is achieved with the use of nucleic acid polymerases, including DNA or RNA polymerases, reverse transcriptase, and Qβ-replicase; (2) exonuclease hydrolysis inhibition which results in only the highest affinity ligands amplifying during PCR. This is achieved with the use of any 3′→5′ double-stranded exonuclease; (3) linear to circle formation to generate molecules amplifiable during PCR; or (4) PCR amplification of single-stranded nucleic acids. A central theme of the method of the present invention is that the nucleic acid candidate mixture is screened in solution and results in preferential amplification of the highest affinity RNA ligand or catalytic RNA.

[0001] This application is a Divisional of U.S. patent application Ser.No. 09/459,553, filed Dec. 13, 1999, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution Selex,” which is a Divisional of U.S. patentapplication Ser. No. 09/093,293, filed Jun. 8, 1998, now U.S. Pat. No.6,001,577, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Photoselection of Nucleic Acid Ligands and Solution Selex,”which is a Continuation of U.S. patent application Ser. No. 08/612,895,filed Mar. 8, 1996, now U.S. Pat. No. 5,763,177, which is a 35 U.S.C.§371 filing of PCT/US94/10562 (WO 95/08003), filed Sep. 16, 1994,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution Selex,” which is aContinuation-in-Part of U.S. patent application Ser. No. 08/123,935,filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,”now abandoned in favor of U.S. patent application Ser. No. 08/443,959,filed May 18, 1995, now abandoned, and a Continuation-in-Part of U.S.patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled“Systematic Evolution of Ligands by Exponential Enrichment: SolutionSELEX,” abandoned in favor of U.S. patent application Ser. No.08/461,069, filed Jun. 5, 1995, now U.S. Pat. No. 5,567,588. U.S. patentapplication Ser. Nos. 08/123,935 and 08/143,564 areContinuations-in-Part of U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096, which is a Continuation-in-Part of U.S. patent applicationSer. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolutionof Ligands by EXponential Enrichment,” now abandoned. U.S. patentapplication Ser. No. 08/143,564 is also a continuation-in-part of U.S.patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled“Methods for Identifying Nucleic Acid Ligands,” now U.S. Pat. No.5,270,163, which is a divisional of U.S. patent application Ser. No.07/714,131.

[0002] This work was supported by grants from the United StatesGovernment funded through the National Institutes of Health. The U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates, in part, to a method for selectingnucleic acid ligands which bind and/or photocrosslink to and/orphotoinactivate a target molecule. The target molecule may be a protein,pathogen or toxic substance, or any biological effector. The nucleicacid ligands of the present invention contain photoreactive orchemically reactive groups and are useful, inter alia, for the diagnosisand/or treatment of diseases or pathological or toxic states.

[0004] The underlying method utilized in this invention is termed SELEX,an acronym for Systematic Evolution of Ligands by EXponentialenrichment. An improvement of the SELEX method herein described, termedSolution SELEX, allows more efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule. Animprovement of the high affinity nucleic acid products of SELEX areuseful for any purpose to which a binding reaction may be put, forexample in assay methods, diagnostic procedures, cell sorting, asinhibitors of target molecule function, as therapeutic agents, asprobes, as sequestering agents and the like.

BACKGROUND OF THE INVENTION

[0005] The SELEX method (hereinafter termed SELEX), described in U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled“Systematic Evolution of Ligands By Exponential Enrichment,” nowabandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10,1991, entitled “Nucleic Acid Ligands,” issued as U.S. Pat. No. 5,475,096and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992,entitled “Methods for Identifying Nucleic Acid Ligands,” issued as U.S.Pat. No. 5,270,163, all of which are herein specifically incorporated byreference (referred to herein as the SELEX Patent Applications),provides a class of products which are nucleic acid molecules, eachhaving a unique sequence, each of which has the property of bindingspecifically to a desired target compound or molecule. Each nucleic acidmolecule is a specific ligand of a given target compound or molecule.SELEX is based on the unique insight that nucleic acids have sufficientcapacity for forming a variety of two- and three-dimensional structuresand sufficient chemical versatility available within their monomers toact as ligands (form specific binding pairs) with virtually any chemicalcompound, whether monomeric or polymeric. Molecules of any size canserve as targets.

[0006] The SELEX method involves selection from a mixture of candidatesand step-wise iterations of structural improvement, using the samegeneral selection theme, to achieve virtually any desired criterion ofbinding affinity and selectivity. Starting from a mixture of nucleicacids, preferably comprising a segment of randomized sequence, themethod includes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound to target molecules,dissociating the nucleic acid-target pairs, amplifying the nucleic acidsdissociated from the nucleic acid-target pairs to yield aligand-enriched mixture of nucleic acids, then reiterating the steps ofbinding, partitioning, dissociating and amplifying through as manycycles as desired.

[0007] While not bound by theory, SELEX is based on the inventors'insight that within a nucleic acid mixture containing a large number ofpossible sequences and structures there is a wide range of bindingaffinities for a given target. A nucleic acid mixture comprising, forexample a 20 nucleotide randomized segment can have 4²⁰ candidatepossibilities. Those which have the higher affinity constants for thetarget are most likely to bind to the target. After partitioning,dissociation and amplification, a second nucleic acid mixture isgenerated, enriched for the higher binding affinity candidates.Additional rounds of selection progressively favor the best ligandsuntil the resulting nucleic acid mixture is predominantly composed ofonly one or a few sequences. These can then be cloned, sequenced andindividually tested for binding affinity as pure ligands.

[0008] Cycles of selection, partition and amplification are repeateduntil a desired goal is achieved. In the most general case,selection/partition/amplification is continued until no significantimprovement in binding strength is achieved on repetition of the cycle.The method may be used to sample as many as about 10¹⁸ different nucleicacid species. The nucleic acids of the test mixture preferably include arandomized sequence portion as well as conserved sequences necessary forefficient amplification. Nucleic acid sequence variants can be producedin a number of ways including synthesis of randomized nucleic acidsequences and size selection from randomly cleaved cellular nucleicacids. The variable sequence portion may contain fully or partiallyrandom sequence; it may also contain subportions of conserved sequenceincorporated with randomized sequence. Sequence variation in testnucleic acids can be introduced or increased by mutagenesis before orduring the selection/partition/amplification iterations.

[0009] Photocrosslinking of nucleic acids to proteins has been achievedthrough incorporation of photoreactive functional groups in the nucleicacid. Photoreactive groups which have been incorporated into nucleicacids for the purpose of photocrosslinking the nucleic acid to anassociated protein include 5-bromouracil, 4-thiouracil, 5-azidouracil,and 8-azidoadenine (see FIG. 1).

[0010] Bromouracil has been incorporated into both DNA and RNA bysubstitution of bromodeoxyuracil (BrdU) and bromouracil (BrU) forthymine and uracil, respectively. BrU-RNA has been prepared with5-bromouridine triphosphate in place of uracil using T7 RNA polymeraseand a DNA template, and both BrU-RNA and BrdU-DNA have been preparedwith 5-bromouracil and 5-bromodeoxyuracil phosphoramidites,respectively, in standard nucleic acid synthesis (Talbot et al. (1990)Nucleic Acids Res. 18:3521). Some examples of the photocrosslinking ofBrdU-substituted DNA to associated proteins are as follows:BrdU-substituted DNA to proteins in intact cells (Weintraub (1973) ColdSpring Harbor Symp. Quant. Biol. 38:247); BrdU-substituted lac operatorDNA to lac repressor (Lin and Riggs (1974) Proc. Natl. Acad. Sci. U.S.A.71:947; Ogata and Gilbért (1977) Proc. Natl. Acad. Sci. U.S.A. 74:4973;Barbier et al. (1984) Biochemistry 23:2933; Wick and Matthews (1991) J.Biol. Chem. 266:6106); BrdU-substituted DNA to EcoRI and EcoRVrestriction endonucleases (Wolfes et al. (1986) Eur. J. Biochem.159:267); Escherichia coli BrdU-substituted DNA to cyclic adenosine3′,5′-monophosphate receptor protein (Katouzian-Safadi et al. (1991)Photochem. Photobiol. 53:611); BrdU-substituted DNA oligonucleotide ofhuman polyomavirus to proteins from human fetal brain extract (Khaliliet al. (1988) EMBO J. 7:1205); a yeast BrdU-substituted DNAoligonucleotide to GCN4, a yeast transcriptional activator (Blatter etal. (1992) Nature 359:650); and a BrdU-substituted DNA oligonucleotideof Methanosarcina sp CHT155 to the chromosomal protein Mc1(Katouzian-Safadi et al. (1991) Nucleic Acids Res. 19:4937).Photocrosslinking of BrU-substituted RNA to associated proteins has alsobeen reported: BrU-substituted yeast precursor tRNA^(Phe) to yeast tRNAligase (Tanner et al. (1988) Biochemistry 27:8852) and a BrU-substitutedhairpin RNA of the R17 bacteriophage genome to R17 coat protein (Gott etal. (1991) Biochemistry 30:6290).

[0011] 4-Thiouracil-substituted RNA has been used to photocrosslink,especially, t-RNA's to various associated proteins (Favre (1990) in:Bioorganic Photochemistry, Volume 1: Photochemistry and the NucleicAcids, H. Morrison (ed.), John Wiley & Sons: New York, pp. 379-425;Tanner et al. (1988) supra). 4-Thiouracil has been incorporated into RNAusing 4-thiouridine triphosphate and T7 RNA polymerase or using nucleicacid synthesis with the appropriate phosphoramidite; it has also beenincorporated directly into RNA by exchange of the amino group ofcytosine for a thiol group with hydrogen sulfide. Yet another method ofsite specific incorporation of photoreactive groups into nucleic acidsinvolves use of 4-thiouridylyl-(3′-5′)-guanosine (Wyatt et al. (1992)Genes & Development 6:2542).

[0012] Examples of 5-azidouracil-substituted and8-azidoadenine-substituted nucleic acid photocrosslinking to associatedproteins are also known. Associated proteins that have been crosslinkedinclude terminal deoxynucleotidyl transferase (Evans et al. (1989)Biochemistry 28:713; Farrar et al. (1991) Biochemistry 30:3075); XenopusTFIIIA, a zinc finger protein (Lee et al. (1991) J. Biol. Chem.266:16478); and E. coli ribosomal proteins (Wower et al. (1988)Biochemistry 27:8114). 5-Azidouracil and 8-azidoadenine have beenincorporated into DNA using DNA polymerase or terminal transferase.Proteins have also been photochemically labelled by exciting8-azidoadenosine 3′,5′-biphosphate bound to bovine pancreaticribonuclease A (Wower et al. (1989) Biochemistry 28:1563) and8-azidoadenosine 5′-triphosphate bound to ribulose-bisphosphatecarboxylase/oxygenase (Salvucci and Haley (1990) Planta 181:287).

[0013] 8-Bromo-2′-deoxyadenosine as a potential photoreactive group hasbeen incorporated into DNA via the phosphoramidite (Liu and Verdine(1992) Tetrahedron Lett. 33:4265). The photochemical reactivity has yetto be investigated.

[0014] Photocrosslinking of 5-iodouracil-substituted nucleic acids toassociated proteins has not been previously investigated, probablybecause the size of the iodo group has been thought to preclude specificbinding of the nucleic acid to the protein of interest. However,5-iodo-2′-deoxyuracil and 5-iodo-2′-deoxyuridine triphosphate have beenshown to undergo photocoupling to thymidine kinase from E. coli (Chenand Prusoff (1977) Biochemistry 16:3310).

[0015] Mechanistic studies of the photochemical reactivity of the5-bromouracil chromophore have been reported including studies withregard to photocrosslinking. Most importantly, BrU shows wavelengthdependent photochemistry. Irradiation in the region of 310 nm populatesan n,π* singlet state which decays to ground state and intersystemcrosses to the lowest energy triplet state (Dietz et al. (1987) J. Am.Chem. Soc. 109:1793), most likely the π,π* triplet (Rothman and Kearns(1967) Photochem. Photobiol. 6:775). The triplet state reacts withelectron-rich amino acid residues via initial electron transfer followedby covalent bond formation. Photocrosslinking of triplet 5-bromouracilto the electron rich aromatic amino acid residues tyrosine, tryptophanand histidine (Ito et al. (1980) J. Am. Chem. Soc. 102:7535; Dietz andKoch (1987) Photochem. Photobiol. 46:971), and the disulfide bearingamino acid, cystine (Dietz and Koch (1989) Photochem. Photobiol.49:121), has been demonstrated in model studies. Even the peptidelinkage is a potential functional group for photocrosslinking to tripletBrU (Dietz et al. (1987) supra). Wavelengths somewhat shorter than 308nm populate both the n,π* and π,π* singlet states. The π,π* singletundergoes carbon-bromine bond homolysis as well as intersystem crossingto the triplet manifold (Dietz et al. (1987) supra); intersystemcrossing may occur in part via internal conversion to the n,π* singletstate. Carbon-bromine bond homolysis likely leads to nucleic acid strandbreaks (Hutchinson and Köhnlein (1980) Prog. Subcell. Biol. 7:1; Shetlar(1980) Photochem. Photobiol. Rev. 5:105; Saito and Sugiyama (1990) in:Bioorganic Photochemistry, Volume 1: Photochemistry and the NucleicAcids, H. Morrison, ed., John Wiley and Sons, New York, pp. 317-378).The wavelength dependent photochemistry is outlined in the JablonskiDiagram in FIG. 2 and the model photocrosslinking reactions are shown inFIG. 3.

[0016] The location of photocrosslinks from irradiation of someBrU-substituted nucleoprotein complexes have been investigated. In thelac repressor-BrdU-lac operator complex a crosslink to tyrosine-17 hasbeen established (Allen et al. (1991) J. Biol. Chem. 266:6113). In thearchaebacterial chromosomal protein MC1-BrdU-DNA complex a crosslink totryptophan-74 has been implicated. In yeast BrdU-substituted DNA-GCN4yeast transcriptional activator a crosslink to alanine-238 was reported(Blatter et al. (1992) supra). In this latter example the nucleoproteincomplex was irradiated at 254 nm which populated initially the π,π*singlet state.

[0017] The results of some reactivity and mechanistic studies of5-iodouracil, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxyuracil-substitutedDNA, and 5-iodo-2′-deoxycytosine-substituted DNA have been reported.5-Iodouracil and 5-iodo-2′-deoxyuracil couple at the 5-position toallylsilanes upon irradiation in acetonitrile-water bearing excesssilane with emission from a medium pressure mercury lamp filteredthrough Pyrex glass; the mechanism was proposed to proceed throughinitial carbon-iodine bond homolysis followed by radical addition to theπ-bond of the allylsilane (Saito et al. (1986) J. Org. Chem. 51:5148).

[0018] Aerobic and anaerobic photo-deiodination of5-iodo-2′-deoxyuracil-substituted DNA has been studied as a function ofexcitation wavelength; the intrinsic quantum yield drops by a factor of4 with irradiation in the region of 313 nm relative to the quantum yieldwith irradiation in the region of 240 nm. At all wavelengths themechanism is proposed to involve initial carbon-iodine bond homolysis(Rahn and Sellin (1982) Photochem. Photobiol. 35:459). Similarly,carbon-iodine bond homolysis is proposed to occur upon irradiation of5-iodo-2′-deoxycytidine-substituted DNA at 313 nm (Rahn and Stafford(1979) Photochem. Photobiol. 30:449). Strictly monochromatic light wasnot used in any of these studies. Recently, a 5-iodouracil-substitutedduplex DNA was shown to undergo a photochemical single strand break(Sugiyama et al. (1993) J. Am. Chem. Soc. 115:4443).

[0019] Also of importance with respect to the present invention is theobserved direct population of the triplet states of 5-bromouracil and5-iodouracil from irradiation of the respective S_(O)→T absorption bandsin the region of 350-400 nm (Rothman and Kearns (1967) supra).

[0020] Photophysical studies of the 4-thiouracil chromophore implicatethe π,π* triplet state as the reactive state. The intersystem crossingquantum yield is unity or close to unity. Although photocrosslinkingwithin 4-thiouracil-substituted nucleoprotein complexes has beenobserved, amino acid residues reactive with excited 4-thiouracil havenot been established (Favre (1990) supra). The addition of the α-aminogroup of lysine to excited 4-thiouracil at the 6-position has beenreported; however, this reaction is not expected to be important inphotocrosslinking within nucleoprotein complexes because the α-aminogroup is involved in a peptide bond (Ito et al. (1980) Photochem.Photobiol. 32:683).

[0021] Photocrosslinking of azide-bearing nucleotides or nucleic acidsto associated proteins is thought to proceed via formation of thesinglet and/or triplet nitrene (Bayley and Knowles (1977) MethodsEnzymol. 46:69; Czarnecki et al. (1979) Methods Enzymol. 56:642; Hannaet al. (1993) Nucleic Acids Res. 21:2073). Covalent bond formationresults from insertion of the nitrene in an O—H, N—H, S—H or C—H bond.Singlet nitrenes preferentially insert in heteroatom-H bonds and tripletnitrenes in C—H bonds. Singlet nitrenes can also rearrange to azirineswhich are prone to nucleophilic addition reactions. If a nucleophilicsite of a protein is adjacent, crosslinking can also occur via thispathway. A potential problem with the use of an azide functional groupresults if it resides ortho to a ring nitrogen; the azide will exist inequilibrium with a tetrazole which is much less photoreactive.

[0022] The coat protein-RNA hairpin complex of the R17 bacteriophage isan ideal system for the study of nucleic acid-protein photocrosslinkingbecause of the simplicity of the system in vitro. The system is wellcharacterized, consisting of a viral coat protein that binds with highaffinity to an RNA hairpin within the phage genome. In vivo theinteraction of the coat protein with the RNA hairpin plays two rolesduring phage infection: the coat protein acts as a translationalrepressor of replicase synthesis (Eggen and Nathans (1969) J. Mol. Biol.39:293), and the complex serves as a nucleation site for encapsidation(Ling et al. (1970) Virology 40:920; Beckett et al. (1988) J. Mol Biol.204:939). Many variations of the wild-type hairpin sequence also bind tothe coat protein with high affinity (Tuerk & Gold (1990) Science249:505; Gott et al. (1991) Biochemistry 30:6290; Schneider et al.(1992) J. Mol. Biol. 228:862).

[0023] The selection of nucleic acid ligands according to the SELEXmethod may be accomplished in a variety of ways, such as on the basis ofphysical characteristics. Selection on the basis of physicalcharacteristics may include physical structure, electrophoreticmobility, solubility, and partitioning behavior. U.S. patent applicationSer. No. 07/960,093, filed Oct. 14, 1992, entitled “Method for SelectingNucleic Acids on the Basis of Structure,” now abandoned (See, U.S. Pat.No. 5,707,796) herein specifically incorporated by reference, describesthe selection of nucleic acid sequences on the basis of specificelectrophoretic behavior. The SELEX technology may also be used inconjunction with other selection techniques, such as HPLC, columnchromatography, chromatographic methods in general, solubility in aparticular solvent, or partitioning between two phases.

BRIEF SUMMARY OF THE INVENTION

[0024] In one embodiment, the present invention includes a method forselecting and identifying nucleic acid ligands from a candidate mixtureof randomized nucleic acid sequences on the basis of the ability of therandomized nucleic acid sequences to bind and/or photocrosslink to atarget molecule. This embodiment is termed Covalent SELEX generally, andPhotoSELEX specifically when irradiation is required to form covalentlinkage between the nucleic acid ligand and the target.

[0025] In one variation of this embodiment, the method comprisespreparing a candidate mixture of nucleic acid sequences which containphotoreactive groups; contacting the candidate mixture with a targetmolecule wherein nucleic acid sequences having increased affinity to thetarget molecule bind the target molecule, forming nucleic acid-targetmolecule complexes; irradiating the nucleic acid-target moleculemixture, wherein some nucleic acids incorporated in nucleic acid-targetmolecule complexes crosslink to the target molecule via thephotoreactive functional groups; taking advantage of the covalent bondto partition the crosslinked nucleic acid-target molecule complexes fromfree nucleic acids in the candidate mixture; and identifying the nucleicacid sequences that were photocrosslinked to the target molecule. Theprocess can further include the iterative step of amplifying the nucleicacids that photocrosslinked to the target molecule to yield a mixture ofnucleic acids enriched in sequences that are able to photocrosslink tothe target molecule.

[0026] In another variation of this embodiment of the present invention,nucleic acid ligands to a target molecule selected through SELEX arefurther selected for their ability to crosslink to the target. Nucleicacid ligands to a target molecule not containing photoreactive groupsare initially identified through the SELEX method. Photoreactive groupsare then incorporated into these selected nucleic acid ligands, and theligands contacted with the target molecule. The nucleic acid-targetmolecule complexes are irradiated and those able to photocrosslink tothe target molecule identified.

[0027] In another variation of this embodiment of the present invention,photoreactive groups are incorporated into all possible positions in thenucleic acid sequences of the candidate mixture. For example,5-iodouracil and 5-iodocytosine may be substituted at all uracil andcytosine positions. The first selection round is performed withirradiation of the nucleic acid-target molecule complexes such thatselection occurs for those nucleic acid sequences able to photocrosslinkto the target molecule. Then SELEX is performed with the nucleic acidsequences able to photocrosslink to the target molecule to selectcrosslinking sequences best able to bind the target molecule.

[0028] In another variation of this embodiment of the present invention,nucleic acid sequences containing photoreactive groups are selectedthrough SELEX for a number of rounds in the absence of irradiation,resulting in a candidate mixture with a partially enhanced affinity forthe target molecule. PhotoSELEX is then conducted with irradiation toselect ligands able to photocrosslink to the target molecule.

[0029] In another variation of this embodiment of the present invention,SELEX is carried out to completion with nucleic acid sequences notcontaining photoreactive groups, and nucleic acid ligands to the targetmolecule selected. Based on the sequences of the selected ligands, afamily of related nucleic acid sequences is generated which contain asingle photoreactive group at each nucleotide position. PhotoSELEX isperformed to select a nucleic acid ligand capable of photocrosslinkingto the target molecule.

[0030] In a further variation of this embodiment of the presentinvention, a nucleic acid ligand capable of modifying the bioactivity ofa target molecule through binding and/or crosslinking to a targetmolecule is selected through SELEX, photoSELEX, or a combination ofthese methods.

[0031] In a further variation of this embodiment of the presentinvention, a nucleic acid ligand to a unique target molecule associatedwith a specific disease process is identified. In yet another variationof this embodiment of the present invention, a nucleic acid ligand to atarget molecule associated with a disease state is used to treat thedisease in vivo.

[0032] The present invention further encompasses nucleic acid sequencescontaining photoreactive groups. The nucleic acid sequences may containsingle or multiple photoreactive groups. Further, the photoreactivegroups may be the same or different in a single nucleic acid sequence.The photoreactive groups incorporated into the nucleic acids of theinvention include any chemical group capable of forming a crosslink witha target molecule upon irradiation. Although in some cases irradiationmay not be necessary for crosslinking to occur.

[0033] The nucleic acids of the present invention include single- anddouble-stranded RNA and single- and double-stranded DNA. The nucleicacids of the present invention may contain modified groups such as2′-amino (2′-NH₂) or 2′-fluoro (2′-F)-modified nucleotides. The nucleicacids of the present invention may further include backbonemodifications.

[0034] The present invention further includes the method wherebycandidate mixtures containing modified nucleic acids are prepared andutilized in the SELEX process, and nucleic acid ligands are identifiedthat bind or crosslink to the target species. In one example of thisembodiment, the candidate mixture is comprised of nucleic acids whereinall uracil residues are replaced by 5-halogenated uracil residues, andnucleic acid ligands are identified that form covalent attachments tothe selected target.

[0035] An additional embodiment of the present invention, termedsolution SELEX, presents several improved methods for partitioningbetween ligands having high and low affinity nucleic acid-targetcomplexes is achieved in solution and without, or prior to, use of apartitioning matrix. Generally, a central theme of the method ofsolution SELEX is that the nucleic acid candidate mixture is treated insolution and results in preferential amplification during PCR of thehighest affinity nucleic acid ligands or catalytic RNAs. The solutionSELEX method achieves partitioning between high and low affinity nucleicacid-target complexes through a number of methods, including (1) Primerextension inhibition which results in differentiable cDNA products suchthat the highest affinity ligands may be selectively amplified duringPCR. Primer extension inhibition is achieved with the use of nucleicacid polymerases, including DNA or RNA polymerases, reversetranscriptase, and Qβ-replicase. (2) Exonuclease hydrolysis inhibitionwhich also results in only the highest affinity ligands amplifyingduring PCR. This is achieved with the use of any 3′→5′ double-strandedexonuclease. (3) Linear to circle formation to generate differentiablecDNA molecules resulting in amplification of only the highest affinityligands during PCR.

[0036] In one embodiment of the solution SELEX method, synthesis ofcDNAs corresponding to low affinity oligonucleotides are preferentiallyblocked and thus rendered non-amplifiable by PCR. In another embodiment,low affinity oligonucleotides are preferentially removed by affinitycolumn chromatography prior to PCR amplification. Alternatively, highaffinity oligonucleotides may be preferentially removed by affinitycolumn chromatography. In yet another embodiment of the SELEX method,cDNAs corresponding to high affinity oligonucleotides are preferentiallyrendered resistant to nuclease enzyme digestion. In a furtherembodiment, cDNAs corresponding to low affinity oligonucleotides arerendered preferentially enzymatically or chemically degradable.

[0037] Solution SELEX is an improvement over prior art partitioningschemes. With the method of the present invention, partitioning isachieved without inadvertently also selecting ligands that only haveaffinity for the partitioning matrix, the speed and accuracy ofpartitioning is increased, and the procedure may be readily automated.

[0038] The present disclosure provides non-limiting examples which areillustrative and exemplary of the invention. Other partitioning schemesand methods of selecting nucleic acid ligands through binding andphotocrosslinking to a target molecule will be become apparent to oneskilled in the art from the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0039]FIG. 1 shows structures of photoreactive chromophores which havebeen incorporated into nucleic acids.

[0040]FIG. 2 shows a Jablonski energy level diagram for the5-bromouracil chromophore and the reactivity of the various excitedstates.

[0041]FIG. 3 shows the model reactions for photocrosslinking of the5-bromouracil chromophore to amino acid residues such as tyrosine,tryptophan, histidine, and cystine.

[0042]FIG. 4 compares UV absorption by thymidine, 5-bromouracil,5-iodouracil, and L-tryptophan in TMK pH 8.5 buffer (100 mMtris(hydroxymethyl)aminomethane hydrochloride, 10 mM magnesium acetate,and 80 mM potassium chloride). The emission wavelengths of the XeCl andHeCd lasers are also indicated. Of particular importance is absorptionby 5-iodouracil at 325 nm without absorption by tryptophan or thymidine.The molar extinction coefficient for 5-iodouracil at 325 nm is 163 L/molcm.

[0043]FIG. 5 shows structures of photoreactive chromophores which can beincorporated into randomized nucleic acid sequences.

[0044]FIG. 6 shows the structures of hairpin RNA sequences RNA-1 (SEQ IDNO:1), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3) containing5-bromouracil, 5-iodouracil, and uracil, respectively. These arevariants of the wild-type hairpin of the R17 bacteriophage genome whichbind tightly to the R17 coat protein.

[0045]FIG. 7 shows binding curves for RNA-1 (SEQ ID NO:1), RNA-2 (SEQ IDNO:2), and RNA-3 (SEQ ID NO:3) to R17 coat protein. The bindingconstants calculated from the binding curves are also given.

[0046]FIG. 8 shows the percent of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ IDNO:2) photocrosslinked to R17 coat protein with monochromaticirradiation at 308 nm from a XeCl excimer laser as a function of time.Photocrosslinking of RNA-1 (SEQ ID NO:1) maximized at 40% because ofcompetitive photodamage to the coat protein during the irradiationperiod. Less photodamage to coat protein occurred with RNA-2 (SEQ IDNO:2) because of the shorter irradiation time.

[0047]FIG. 9 shows the percent of RNA-2 (SEQ ID NO:2) photocrosslinkedto R17 coat protein with monochromatic irradiation at 325 nm from a HeCdlaser as a function of time. The data are also presented in the originalelectrophoretic gel format. The symbol IU XL marks RNA crosslinked toprotein. A near-quantitative yield of photocrosslinking was obtained.

[0048]FIG. 10 shows the percent of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ IDNO:2) photocrosslinked to R17 coat protein with broad-band irradiationin the region of 312 nm from a Transilluminator as a function of time.Less than quantitative yields of photocrosslinking were obtained becauseof photodamage to the protein and possibly to the RNA sequences.

[0049]FIG. 11 shows formation of the same product, Structure 6, fromirradiation at 308 nm of 5-iodouracil and 5-bromouracil in the presenceof excess N-acetyltyrosine N-ethylamide (Structure 5).

[0050]FIG. 12 pictures photocrosslinking of RNA-7 (SEQ ID NO:4) to R17coat protein with 308 nm light followed by enzymatic digestion of mostof the coat protein.

[0051]FIG. 13 shows formation of a complementary DNA from the RNAtemplate after enzymatic digestion of the coat protein of FIG. 12 (SEQID NO:4).

[0052]FIG. 14 shows the polyacrylamide gel of Example 8 showingproduction of a cDNA from an RNA template bearing modified nucleotidesas shown in FIGS. 12 and 13. The modified nucleotides were 5-iodouraciland uracil substituted at the 5-position with a small peptide. Basedupon model studies shown in FIG. 11, the peptide was most likelyattached to the uracil via the phenolic ring of a tyrosine residue.

[0053]FIG. 15 shows the photocrosslinking of [α-³²P] GTP labelled poolRNA to HIV-1 Rev protein using tRNA competition.

[0054]FIG. 16 (SEQ ID NOS:5-55) shows the sequence of the previouslyidentified RNA ligand to HIV-1 Rev protein that is referred to herein as6a (SEQ ID NO:5). Also shown are 52 sequences from round 13 selected forphotocrosslinking to HIV-1 Rev protein.

[0055]FIG. 17 (SEQ ID NOS:56-57) shows the consensus for class 1 ligandsand class 2 ligands. Class 1: Consensus secondary structure for class 1and class 2 molecules. N₁—N₁′ indicate 1-2 complementary base pairs;N₂—N₂′ indicates 1-4 complementary base pairs, D-H′ is an A-U, U-A, orG-C base pair; K-M′ is a G-C or U-A base pair (16). Class 2: Boldsequences represent the highly conserved 10 nucleotides thatcharacterize class 2 molecules; base-pairing is with the 5′ fixed end ofthe molecule.

[0056]FIG. 18 (SEQ ID NO:58) shows the sequence and predicted secondarystructure of trunc2 (18A). Also shown (18B) is a gel demonstrating thespecificity of trunc2 photocrosslinking to ARM proteins.

[0057]FIG. 19 shows the sequence and predicted secondary structure oftrunc24 (SEQ ID NO:59) (19A). Also shown (19B) is a gel demonstratingthe specificity of laser independent crosslinking to ARM proteins.

[0058]FIG. 20 shows the trunc24 (SEQ ID NO:59) photo-independentcrosslinking with HIV-1 Rev in the presence of human nuclear extracts.

[0059]FIG. 21 illustrates the cyclical relationship between SELEX steps.A single-stranded nucleic acid repertoire of candidate oligonucleotidesis generated by established procedures on a nucleic acid synthesizer,and is amplified by PCR to generate a population of double-stranded DNAmolecules. Candidate DNA or RNA molecules are generated throughasymmetric PCR or transcription, respectively, purified, and allowed tocomplex with a target molecule. This is followed by partitioning ofbound and unbound nucleic acids, synthesis of cDNA, and PCRamplification to generate double-stranded DNA.

[0060]FIG. 22 illustrates one embodiment of the solution SELEX method inwhich primer extension inhibition is used to create differentiable cDNApools—an amplifiable high affinity oligonucleotide cDNA pool and anon-amplifiable low affinity oligonucleotide cDNA pool. In thisembodiment, the first cDNA extension is performed in the presence ofchain terminating nucleotides such as ddG. After removal of the targetmolecule and dideoxynucleotides, the second cDNA extension is conductedin the presence of four dNTPs. Full-length cDNA is preferentiallysynthesized from the high affinity oligonucleotides and therefore, thehigh affinity cDNA pool is amplified in the subsequent PCR step.

[0061]FIG. 23 illustrates the cyclic solution SELEX process for theembodiment shown in FIG. 22.

[0062]FIG. 24 illustrates one embodiment of the cyclic solution SELEXprocess wherein partitioning between oligonucleotides having high andlow affinity to a target molecule is achieved by restriction enzymedigestion. In this embodiment, the first cDNA extension is conductedwith four dNTPs and results in cDNAs corresponding to the low affinityoligonucleotides. The target is then removed and a second cDNA extensionis conducted in the presence of modified nucleotides resistant toenzymatic cleavage. The cDNA pools are then incubated with restrictionenzyme and only the cDNA pool corresponding to high affinityoligonucleotides is amplifiable in the subsequent PCR step.

[0063]FIG. 25 illustrates one embodiment of the cyclic solution SELEXprocess wherein partitioning between oligonucleotides having high andlow affinity to a target molecule is achieved by affinitychromatography. The first cDNA extension is performed in the presence ofa modified nucleotide such as a biotinylated nucleotide, which allowsthe cDNA pool corresponding to the low-affinity oligonucleotide to beretained on an affinity column.

[0064]FIG. 26 illustrates one embodiment of the solution SELEX processwherein partitioning between oligonucleotides having high and lowaffinity to a target molecule is achieved by exonuclease inhibition andresults in formation of a double-stranded nucleic acid population withhigh affinity for the target molecule.

[0065]FIG. 27 illustrates one embodiment of the solution SELEX processwherein catalytic nucleic acids are selected and isolated.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention includes a variation of the SELEX methodfor selecting nucleic acid ligands. This application hereby specificallyincorporates by reference the full text including the definitionsprovided in the earlier SELEX patent applications, specifically thoseprovided in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, now abandoned, and 07/714,131, filed Jun. 10, 1991, now U.S. Pat.No. 5,475,096. The method of one embodiment of the present invention,termed covalent SELEX or photoSELEX, identifies and selects nucleic acidligands capable of binding and/or photocrosslinking to target molecules.

[0067] This application also presents a method for improved partitioningof nucleic acid ligands identified through the SELEX method.

[0068] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0069] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: a) to assistin the amplification steps described below; b) to mimic a sequence knownto bind to the target; or c) to enhance the potential of a givenstructural arrangement of the nucleic acids in the candidate mixture.The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0070] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthe nucleic acids having the strongest affinity for the target.

[0071] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-10%) is retainedduring partitioning.

[0072] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0073] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acidmixture to the target will generally increase. Taken to its extreme, theSELEX process will yield a candidate mixture containing one or a smallnumber of unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0074] The SELEX Patent Applications describe and elaborate on thisprocess in great detail. Included are targets that can be used in theprocess; methods for the preparation of the initial candidate mixture;methods for partitioning nucleic acids within a candidate mixture; andmethods for amplifying partitioned nucleic acids to generate enrichedcandidate mixtures. The SELEX Patent Applications also describe ligandsolutions obtained to a number of target species, including both proteintargets wherein the protein is and is not a nucleic acid bindingprotein.

[0075] Partitioning means any process whereby ligands bound to targetmolecules can be separated from nucleic acids not bound to targetmolecules. More broadly stated, partitioning allows for the separationof all the nucleic acids in a candidate mixture into at least two poolsbased on their relative affinity to the target molecule. Partitioningcan be accomplished by various methods known in the art. Nucleicacid-protein pairs can be bound to nitrocellulose filters while unboundnucleic acids are not. Columns which specifically retain nucleicacid-target complexes can be used for partitioning. For example,oligonucleotides able to associate with a target molecule bound on acolumn allow use of column chromatography for separating and isolatingthe highest affinity nucleic acid ligands. Liquid-liquid partitioningcan be used as well as filtration gel retardation, and density gradientcentrifugation.

[0076] I. PhotoSELEX

[0077] The present invention encompasses nucleic acid ligands whichbind, photocrosslink and/or photoinactivate target molecules. Binding asreferred to herein generally refers to the formation of a covalent bondbetween the ligand and the target, although such binding is notnecessarily irreversible. Certain nucleic acid ligands of the presentinvention contain photoreactive groups which are capable ofphotocrosslinking to the target molecule upon irradiation with light.Additional nucleic acid ligands of the present invention are capable ofbond formation with the target in the absence of irradiation.

[0078] In one embodiment, the present invention encompasses nucleic acidligands which are single- or double-stranded RNA or DNAoligonucleotides. The nucleic acid ligands of the present invention maycontain photoreactive groups capable of crosslinking to the selectedtarget molecule when irradiated with light. Further, the presentinvention encompasses nucleic acid ligands containing any modificationthereof. Reference to a photoreactive group herein may simply refer to arelatively simple modification to a natural nucleic acid residue thatconfers increased reactivity or photoreactivity to the nucleic acidresidue. Such modifications include, but are not limited to,modifications at cytosine exocyclic amines, substitution withhalogenated groups, e.g., 5′-bromo- or 5′-iodo-uracil, modification atthe 2′-position, e.g., 2′-amino (2′-NH₂) and 2′-fluoro (2′-F), backbonemodifications, methylations, unusual base-pairing combinations and thelike. For example, the nucleic acid ligands of the present invention mayinclude modified nucleotides such as 2′-NH₂-iodouracil,2′-NH₂-iodocytosine, 2′-NH₂-iodoadenine, 2′-NH₂-bromouracil,2′-NH₂-bromocytosine, and 2′-NH₂-bromoadenine.

[0079] In one embodiment of the photoSELEX method of the presentinvention, a randomized set of nucleic acid sequences containingphotoreactive groups, termed the candidate mixture, is mixed with aquantity of the target molecule and allowed to establish an equilibriumbinding with the target molecule. The nucleic acid-target moleculemixture is then irradiated with light until photocrosslinking iscomplete as indicated by polyacrylamide gel electrophoresis. Only someof those nucleic acids binding tightly to the target molecules willefficiently crosslink with the target.

[0080] The candidate mixture of the present invention is comprised ofnucleic acid sequences with randomized regions including chemicallyreactive or a photoreactive group or groups. Preferably the reactivegroups are modified nucleic acids. The nucleic acids of the candidatemixture preferably include a randomized sequence portion as well asconserved sequences necessary for efficient amplification. The variablesequence portion may contain fully or partially random sequence; it mayalso contain subportions of conserved sequence incorporated within therandomized sequence regions.

[0081] Preferably, each oligonucleotide member of the candidate mixturecontains at least one chemically reactive or photoreactive group.Further, each oligonucleotide member of the candidate mixture may bepartially or fully substituted at each position by modified nucleotidescontaining reactive groups. The candidate mixture may also be comprisedof oligonucleotides containing more than one type of reactive group.

[0082] The target molecules bound and/or photocrosslinked by the nucleicacid ligands of the present invention are commonly proteins and areselected based upon their role in disease and/or toxicity. Examples areenzymes for which an inhibitor is desired or proteins for whichdetection is desired. However, the target molecule may be any compoundof interest for which a ligand is desired. A target molecule can be aprotein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,etc., without limitation.

[0083] A photoreactive group for the purpose of this application ispreferably a modified nucleotide that contains a photochromophore, andthat is capable of photocrosslinking with a target species. Althoughreferred to herein as photoreactive groups, in some cases as describedbelow, irradiation is not necessary for covalent binding to occurbetween the nucleic acid ligand and the target. Preferentially, thephotoreactive group will absorb light in a spectrum of the wavelengththat is not absorbed by the target or the non-modified portions of theoligonucleotide. This invention encompasses, but is not limited to,oligonucleotides containing a photoreactive group selected from thefollowing: 5-bromouracil (BrU), 5-iodouracil (IU), 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine,5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine,5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine,8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine,8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine,8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine (FIG. 5). In one embodiment, thephotoreactive groups are 5-bromouracil (BrU) and 5-iodouracil (IU).

[0084] The photoreactive groups of the present invention are capable offorming bonds with the target species upon irradiation of an associatednucleic acid target pair. The associated pair is referred to herein as anucleoprotein complex, and in some cases irradiation is not required forbond formation to occur. The photocrosslink that typically occurs willbe the formation of a covalent bond between the associated nucleic acidand the target. However, a tight ionic interaction between the nucleicacid and target may also occur upon irradiation.

[0085] In one embodiment, photocrosslinking occurs due toelectromagnetic irradiation. Electromagnetic irradiation includesultraviolet light, visible light, X-ray and gamma ray. 5-Halosubstituted deoxyuracils and deoxycytosines are known to sensitize cellsto ionizing radiation (Szybalski (1974) Cancer Chemother. Rep. 58:539).

[0086] Crosslinking experiments have shown that a precise juxtapositionof either IU or BrU and a tyrosine, tryptophan, or histidine is requiredfor a high yield crosslinking to occur. The present invention takesadvantage of this finding with selection for crosslinking molecules withrandomly incorporated photoreactive groups. In one embodiment of thepresent invention, the photoreactive groups 5-bromouracil (BrU) or5-iodouracil (IU) are incorporated into RNA by T7 polymerasetranscription with the 5-halouridine triphosphate present in place ofuridine triphosphate. Incorporation is achieved by using a mixture of5-halouridine triphosphate and uridine triphosphate or all 5-halouridinetriphosphate. A randomized set of ³²P or ³³P-labeled or unlabeled RNAsequences is obtained from a randomized set of DNA templates,synthesized using standard methodology.

[0087] The randomized set of RNA oligonucleotides containing BrU or IUare mixed with a quantity of a target protein. The photoreactivechromophore is incorporated randomly into RNA as BrU or IU in place ofuracil using standard methodology. The RNA-target protein mixture isirradiated with near ultraviolet light in the range of 300 to 325 nmuntil photocrosslinking is complete. Only those photoreactive groupsadjacent to a reactive amino acid residue in a nucleoprotein complexform a covalent bond to the protein. Excited BrU or IU, returns to theground state unless it is near a reactive functional group such as anoxidizable amino acid residue. Amino acid residues which have beenestablished as being reactive with the lowest triplet state of5-bromouracil include tyrosine, tryptophan, histidine, and cystine (seeFIG. 3). Others of potential reactivity based upon mechanistic studiesare phenylalanine, methionine, cysteine, lysine, and arginine.

[0088] Nucleoprotein complexes which do not form crosslinks may beeasily disrupted by adjusting the reaction medium such as by denaturingwith heat and/or salt. Nucleic acids covalently bound to the protein maybe separated from free nucleic acids on a nitrocellulose filter or byother partitioning methods known to those skilled in the art. Alternatemethods for separating nucleic acids covalently bound to targets fromfree nucleic acids include gel electrophoresis followed byelectroelution, precipitation, differential solubility, andchromatography. To one skilled in the art, the method of choice willdepend at least in part on the target molecule of interest. Thecrosslinked nucleic acids are released from the nitrocellulose filter bydigestion of the protein material with enzymes such as Proteinase K. Atthis point 5-halouracil groups which have photocrosslinked to the targetprotein are bound to a single amino acid or to a short peptide. Theread-through ability of reverse transcriptase is not effected byincorporation of a substituent at the 5-position of uracil becausereverse transcriptase (RT) does not differentiate the 5-position ofuracil from that of thymine. Derivatization of the 5-position has beenused to incorporate groups as large as biotin into RNA molecules. In oneembodiment of the present invention, the target is removed from theselected photocrosslinked nucleic acid by photo or chemicaldissociation.

[0089] Complementary nucleic acid copies of the selected RNA sequencesare prepared with an appropriate primer. The cDNA is amplified with aDNA polymerase and a second primer. 5-Halo-2′-deoxyuracil is notemployed in the DNA synthesis and amplification steps. The amplifiedDNAs are then transcribed into RNA sequences using 5-halouridinetriphosphate in place of uridine triphosphate in the same or differentratio of 5-halouridine to uridine in the candidate mixture.

[0090] For the subsequent round of photoSELEX, the partially selectedRNA sequences are again allowed to complex with a quantity of the targetprotein. Subsequently, the nucleoprotein complexes are irradiated in theregion of 300-325 nm. RNA sequences which have crosslinked to proteinare again separated from RNA sequences which have not crosslinked. cDNAsare prepared and amplified and a third set of RNA sequences containing5-halouracil are prepared. The cycle is continued until it converges toone or several RNA ligands which bind with high affinity andphotocrosslink to the target protein. Shortening of the irradiation timein later cycles can further enhance the selection. The cDNAs of theselected RNA ligands are amplified, gel purified, and sequenced.Alternatively, the RNA sequences can be sequenced directly. The selectedRNA sequences are then transcribed from the appropriate synthesized DNAtemplate, again using 5-halouridine triphosphate in place of uridinetriphosphate (Example 11).

[0091] In another embodiment of the present invention, photoSELEX isperformed on oligonucleotide sequences preselected for their ability tobind the target molecule (Example 12). SELEX is initially performed witholigonucleotides which do not contain photoreactive groups. The RNAligand is transformed into a photoreactive ligand by substitution ofphotoreactive nucleic acid nucleotides at specific sites in the ligand.The photochemically active permutations of the initial ligand may bedeveloped through a number of approaches, such as specific substitutionor partial random incorporation of the photoreactive nucleotides.Specific substitution involves the synthesis of a variety ofoligonucleotides with the position of the photoreactive nucleotidechanged manually. The location of the substitution is directed basedupon the available data on binding of the ligand to the target molecule.For example, substitutions are made to the initial ligand in areas ofthe molecule that are known to interact with the target molecule. Forsubsequent selection rounds, the photoSELEX method is used to select forthe ability to crosslink to the target molecule upon irradiation.

[0092] In another embodiment of the present invention, nucleic acidligands are selected by photoSELEX followed by SELEX. PhotoSELEX isperformed initially with oligonucleotide sequences containingphotoreactive groups. Sequences selected for their ability to crosslinkto the target molecule are then selected for ability to bind the targetmolecule through the SELEX method (Example 13).

[0093] In another embodiment of the present invention, a limitedselection of oligonucleotides through SELEX is followed by selectionthrough photoSELEX (Example 14). The initial SELEX selection rounds areconducted with oligonucleotides containing photoreactive groups. After anumber of SELEX rounds, photoSELEX is conducted to selectoligonucleotides capable of binding the target molecule.

[0094] In yet another embodiment of the present invention, nucleic acidligands identified through SELEX are subjected to limited randomization,followed by selection through photoSELEX (Example 15). SELEX is firstcarried out to completion with nucleic acid sequences not containingphotoreactive groups. The sequence of the nucleic acid ligand is used togenerate a family of oligonucleotides through limited randomization.PhotoSELEX is subsequently performed to select a nucleic acid ligandcapable of photocrosslinking to the target molecule.

[0095] In another embodiment of the present invention, photoSELEX isused to identify a nucleic acid ligand capable of modifying thebiological activity of a target molecule (Example 16).

[0096] In a further embodiment of the present invention, the photoSELEXmethodology is applied diagnostically to identify unique proteinsassociated with specific disease states (Example 17). In yet anotherembodiment of the present invention, nucleic acid ligands capable ofcrosslinking a target molecule associated with a specific diseasecondition are used in vivo to crosslink to the target molecule as amethod of treating the disease condition (Examples 18 and 19).

[0097] In one embodiment of the present invention, RNA ligandsidentified by photoSELEX are used to detect the presence of the targetprotein by binding to the protein and then photocrosslinking to theprotein. Detection may be achieved by incorporating ³²P or ³³P-labelsand detecting material which is retained by a nitrocellulose filter byscintillation counting or detecting material which migrates correctly onan electrophoretic gel with photographic film. Alternatively, photoSELEXcreates a fluorescent chromophore which is detected by fluorescenceemission spectroscopy. Fluorescence emission for the products ofreaction of 5-bromouracil to model peptides (as shown in FIG. 3) hasbeen reported by Dietz and Koch (1987) supra. In another embodiment ofthe invention, a fluorescent label is covalently bound to the RNA anddetected by fluorescence emission spectroscopy. In another embodiment ofthe invention, RNA ligands selected through photoSELEX are used toinhibit the target protein through the same process. In yet anotherembodiment, the photoselected ligand is bound to a support and used tocovalently trap a target.

[0098] In a one embodiment of the invention, 5-iodouracil isincorporated into the RNA sequences of the candidate mixture, and lightin the range of 320-325 nm is used for irradiation. This combinationassures regionselective photocrosslinking of the 5-halouracilchromophore to the target protein without other non-specificphotoreactions. In particular, tryptophan residues of proteins andthymine and uracil bases of nucleic acids are known to be photoreactive.As shown in FIG. 4, 5-iodouracil absorbs at 325 nm but tryptophan andthe standard nucleic acid bases do not. The molar extinction coefficientfor 5-iodouracil at 325 mn is 163 L/mol cm. Monochromatic light in theregion of 320-325 nm is preferably supplied by a frequency doubledtunable dye laser emitting at 320 nm or by a helium cadmium laseremitting at 325 nm.

[0099] In one embodiment of the invention a xenon chloride (XeCl)excimer laser emitting at 308 nm is employed for the photocrosslinkingof 5-iodouracil-bearing RNA sequences to a target protein. With thislaser, a high yield of photocrosslinking of nucleoprotein complexes isachieved within a few minutes of irradiation time.

[0100] In another embodiment of the invention, photocrosslinking of5-iodouracil-bearing RNA sequences to a target protein is achieved withwavelength filtered 313 nm high pressure mercury lamp emission or withlow pressure mercury lamp emission at 254 nm absorbed by a phosphor andre-emitted in the region of 300-325 nm. The latter emission is alsocarefully wavelength filtered to remove 254 nm light not absorbed by thephosphor and light in the region of 290-305 nm which could damage theprotein.

[0101] In a further embodiment of the invention, photocrosslinking ofBrU- or IU-bearing RNA sequences to a target protein is achieved withlight in the region of 350-400 nm which populates directly the tripletstate from the ground state. Monochromatic light from the third harmonicof a Neodymium YAG laser at 355 nm or the first harmonic from a xenonfluoride (XeF) excimer laser at 351 nm may be particularly useful inthis regard.

[0102] In yet another embodiment of the invention the photoreactivenucleotides are incorporated into single stranded DNAs and amplifieddirectly with or without the photoreactive nucleotide triphosphate.

[0103] A. Covalent SELEX and Nucleic Acid Ligands that Bind to HIV-1 RevProtein with and without Irradiation

[0104] The target protein chosen to illustrate photo-SELEX is Rev fromHIV-1. Rev's activity in vivo is derived from its association with theRev-responsive element (RRE), a highly structured region in the HIV-1viral RNA. Previous RNA SELEX experiments of Rev have allowed theisolation of very high affinity RNA ligands. The highest affinityligand, known as “6a,” (SEQ ID NO:5) has a K_(d) of approximately 1 nMand is shown in FIG. 16. The secondary structure of 6a, and itsinteraction with Rev, have been well characterized.

[0105] The construction of the nucleic acid library for photo-SELEX wasbased upon the Rev 6a sequence (SEQ ID NO:5). During the synthesis ofthe deoxyoligonucleotide templates for SELEX, the random region of thetemplate was substituted by a “biased randomization” region, in whichthe ratio of the four input bases was biased in favor of thecorresponding base in the 6a sequence. (Actual ratios were62.5:12.5:12.5:12.5.) For example, if the 6a base for a particularposition is G, then the base input mixture for this synthesis step is62.5% G, and 12.5% of the other three bases.

[0106] The photoreactive uracil analogue 5-iodouracil (iU), which hasbeen used to generate high-yield, region-specific crosslinks betweensingly-substituted iU nucleic acids and protein targets (Willis et al.(1993) Science 262:1255) was used for this example. The iU chromophoreis reactive under long-wavelength ultraviolet radiation, and mayphotocouple to the aromatic amino acids of protein targets by the samemechanism as 5-bromouracil (Dietz et al. (1987) J. Am. Chem. Soc.109:1793). As discussed above, the target for this study is the HIV-1Rev protein, which is necessary for productive infection of the virus(Feinberg et al. (1986) Cell 46:807) and the expression of the viralstructural genes gag, pol and env (Emerman et al. (1989) Cell 57:1155).The interaction of Rev with high affinity RNA ligands is wellcharacterized. A single, high-affinity site within the RRE (the IIBstem) has been identified (Heaphy et al. (1991) Proc. Natl. Acad. Sci.USA 88:7366). In vitro genetic selection experiments have generated RNAligands that bind with high affinity to Rev and have helped determinethe RNA structural elements necessary for Rev:RNA interactions (Bartelet al. (1991) Cell 67:529; Tuerk et al., In the Polymerase ChainReaction (1993); Jensen et al. (1994) J. Mol. Biol. 235:237).

[0107] A “biased randomization” DNA oligonucleotide library, based uponthe high affinity Rev ligand sequence 6a (SEQ ID NO:5), containsapproximately 10¹⁴ unique sequences. This template was used for in vitroT7 transcription with 5-iUTP to generate fully-substituted iU RNA forselection. The photo-SELEX procedure alternated between affinityselection for Rev using nitrocellulose partitioning and monochromatic UVirradiation of the nucleoprotein complexes with denaturingpolyacrylamide gel partitioning of the crosslinked complexes away fromnon-crosslinked RNA sequences. The final procedure utilized asimultaneous selection for affinity and crosslinking using competitortRNA. Each round constitutes a selection followed by the conversion ofrecovered RNA to cDNA, polymerase chain reaction (PCR) amplification ofthe DNA, and in vitro transcription to generate a new pool of iU-RNA. Toamplify RNA's recovered as covalent nucleoprotein complexes, theappropriate gel slice was isolated and proteinase K treated.

[0108] The RNA pool was first subjected to three rounds of affinityselection with Rev protein, with partitioning of the higher affinitysequences by nitrocellulose filters. Next, the evolving RNA pool wassubjected to UV laser irradiation in the presence of excess Rev proteinto allow those RNA sequences with the ability to crosslink with theprotein to do so. Crosslinked RNA sequences were then partitioned usingpolyacrylamide gel electrophoresis (PAGE). These crosslinked RNAs wererecovered from the gel material, the linked Rev protein digested away,and the RNAs used for cDNA synthesis and further amplification for thenext round of photo-SELEX. A 308 nm XeCl excimer laser was used for thefirst round of photocrosslinking; thereafter, a 325 nm HeCd laser wasemployed.

[0109] Following four rounds of selection for laser-inducedcrosslinking, the RNA pool was again put through three rounds ofaffinity selection. Finally, the RNA pool was selected simultaneouslyfor its ability to bind Rev with high affinity and to crosslink to theprotein. This was accomplished by using high concentrations of anon-specific nucleic acid competitor in the photocrosslinking reaction.

[0110] Crosslinked product increased approximately 30-fold from thestarting pool to round 13 (FIG. 15). Under these conditions, thegreatest increase in crosslinking is correlated with the greatestincrease in affinity—from round 7 to round 10.

[0111] After 13 rounds of selection, the PCR products were cloned and 52isolates sequenced (FIG. 16, SEQ ID NOS:5-55). Class 1 molecules, whichcomprise 77% of the total sequences, contain a very highly conservedmotif, 5′KDAACAN . . . N′UGUUH′M′3′ (SEQ ID NO:56) (FIG. 17). Computerfolding algorithms predict that this conserved motif is base-paired andlies in a stem-loop structure. Subclasses a-d (FIG. 16, SEQ ID NOS:5-43)illustrate different strategies utilized in the “biased randomization”pool to obtain the consensus motif. Class 2 molecules show a highlyconserved 10-base sequence (FIG. 17, SEQ ID NO. 57), which is predictedto fold with the 5′ fixed region of the RNA and forms a structuredistinct from either the class 1 or the 6a (SEQ ID NO:5) motif. Allclass 1 sequences exhibit biphasic binding to Rev, with high affinitydissociation constants (K_(d)s) ranging from 1-10 nM. Class 2 sequencesshow monophasic binding to Rev with K_(d)s approximately of 30-50 nM.Analysis of round 13 sequences reveal that the frequency of theconsensus motifs for class 1 and class 2 populations was very small inthe starting pool, and some individual sequences arose only through themutational pressures of the photo-SELEX procedure.

[0112] Cross-linking behavior differs between the two classes. Underhigh Rev concentrations (500 nM), and 4 min. of 325 nm irradiation,class 2 molecules produce greater crosslink yield and efficiency thanclass 1 molecules (data not shown); presumably, this behavior allows theclass 2 molecules, with relatively low affinity for Rev, to competeunder the photo-SELEX procedures. For class 1 molecules, longerirradiation times will produce higher molecular weight crosslinkedspecies. Although not bound by theory, it is proposed that the RNAs,which contain both an evolved binding domain for Rev, and the fixedregions needed for amplification in SELEX, are able to bind more thanone Rev molecule per RNA. Since each RNA contains on average 21 iU bases(RNA length-86 bases), it is thought that there is a certain promiscuityof the photoreaction that allows crosslinking of a single RNA to morethan one Rev molecule at high protein concentrations. Class 2 moleculesproduce fewer high molecular weight species upon photocrosslinking; theyare, on average, iU poor and may contain structures which do not allowbinding/crosslinking to additional Rev molecules.

[0113] Analysis of individual round 13 RNAs revealed that asubpopulation could crosslink to Rev without laser irradiation. Thus,the single set of experiments demonstrated that both covalent SELEXwithout irradiation and photoSELEX with irradiation can be found in thesame system. 4 of 15 round 13 sequences analyzed crosslink without laserirradiation (FIG. 16). From these few sequences, it was not readilypossible to identify a sequence motif that confers laser independentcrosslinking, although all molecules considered to date belong to the 1asubclass.

[0114] To further investigate laser-dependent and laser-independentcrosslinking (LD-XL and LI-XL, respectively) and avoid the secondaryphotoproducts formed with full-length class 1 molecules, several smallRNAs containing only the evolved sequences were constructed. Trunc2 andtrunc24 (FIGS. 18 and 19) (SEQ ID NOS:58 AND 59) are based upon clones#3 and #24, respectively, and show monophasic binding to Rev with Kds of0.5 nM (trunc2) and 20 nM (trunc24). Trunc2 (FIG. 18) exhibits LD-XLbehavior, and trunc24 (FIG. 19) is capable of both LI-XL and LD-XL.

[0115] To explore the conformation and chemical requirements for LD-XLand LI-XL, crosslinking reactions were performed with trunc2 (SEQ IDNO:58) and trunc24 (SEQ ID NO:59) and several Arginine Rich Motif (ARM)proteins. The class of RNA-binding proteins includes the target protein,HIV-1 Rev, and also HIV-1 Tat and the highly similar HIV-2 Rev. LD-XLreactions with trunc2 (FIG. 18, SEQ ID NO:58) show that trunc2 iscapable of crosslinking specifically to both HIV-1 and HIV-2 Revproteins, but not HIV-1 Tat. The two slightly different migratingnucleoprotein complexes probably represent the ability of trunc2 to useone of two iU nucleotides to crosslink the Rev proteins. Although notbound by theory, it is proposed that a tryptophan residue present in thehighly similar ARMs of both Rev proteins is the amino acid necessary forthe specific photo-crosslinking of our high-affinity RNA ligands.

[0116] Trunc24 LI-XL (FIG. 19, SEQ ID NO:59), performed with the sameproteins, shows crosslinking only to HIV-1 Rev. Like trunc3, trunc24 canphoto-crosslink to HIV-2 Rev (data not shown). It was also observed thatthis LI-crosslink is reversible under highly denaturing conditions, orwith high concentration of nucleic acid competitors. Although not boundby theory, these observations lead to the postulation that LI-XLproceeds by a Michael adduct between the 6 position of an IU and acysteine residue, or possibly a 5 position substitution reaction. Thispostulation is consistent both with the observation that iU undergoesMichael adduct formation more readily than U, and the fact that HIV-1Rev contains three cysteines, while HIV-2 Rev contains none.

[0117] To test for the ability of trunc24 (SEQ ID NO:59) to discriminateHIV-1 Rev in a complex mixture, trunc24 and 10 μg of human fibroblastnuclear extract were mixed together with decreasing amounts of HIV-1 Rev(FIG. 20). At 50 nM Rev and a 1:100 weight ratio of Rev to nuclearextract, it was possible to see a very significant crosslinked productbetween trunc24 and Rev. Nuclear extracts and trunc24 alone resulted inno crosslinked products.

[0118] Example 1 describes the synthesis of hairpin RNA oligonucleotidesRNA-1 (SEQ ID NO:1), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3) using5-bromouridine triphosphate, 5-iodouridine triphosphate and uridinetriphosphate, respectively. Experiments determining the RNA-proteinbinding curves for RNA-1 (SEQ ID NO:1), RNA-2 (SEQ ID NO:2), and RNA-3(SEQ ID NO:3) to the bacteriophage R17 coat protein are described inExample 2. Example 3 describes the photocrosslinking of the RNAoligonucleotides to the R17 coat protein. The amino acid residue of theR17 coat protein photocrosslinked by RNA-1 (SEQ ID NO:1) afterillumination via xenon chloride (XeCl) excimer laser at 308 nm isdescribed in Example 4. Example 5 describes the photocrosslinking ofiodouracil-substituted RNA-2 (SEQ ID NO:2) to the R17 coat protein bymonochromatic emission at 325 nm. Example 6 describes thephotocrosslinking of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) to theR17 coat protein achieved after broad-band emission illumination with atransilluminator. Example 7 describes the photoreaction of 5-iodouracilwith N-acetyltyrosine N-ethyl amide, which appeared to yield aphotocrosslink similar to that achieved with 5-bromouracil-substitutednucleic acids to associated proteins. The preparation of a cDNA from aRNA photocrosslinked to the R17 coat protein is described in Example 8.Example 9 describes the photocrosslinking of an IC-substituted RNAligand to the R17 coat protein.

[0119] Example 10 describes the incorporation of halogenated nucleotidesinto DNA. Examples 11-15 describes photoSELEX protocols which may beused to produce specific photoreactive nucleic acid ligands. Example 11describes a continuous photoSELEX method. Example 12 describes a methodin which nucleic acid ligands initially selected through SELEX aresubsequently selected through photoSELEX for the capacity to crosslinkto the target molecule. Example 13 describes one embodiment in whichnucleic acid ligands identified through photoSELEX are then subjected toselection through SELEX and selected for ability to bind the targetmolecule. Example 14 describes another embodiment wherein a limitedSELEX selection is followed by selection through photoSELEX. Example 15describes an embodiment of the present invention in which nucleic acidligands identified through SELEX are subjected to limited randomization,followed by selection through photoSELEX. Example 16 describes a methodfor selecting a nucleic acid ligand capable of modifying the biologicalactivity of a target molecule. Example 17 describes a diagnosticprocedure which uses the SELEX and photoSELEX methods to identifyproteins associated with specific disease processes.

[0120] Example 18 describes a method for the in vivo treatment ofdisease through photoSELEX. A photoSELEX selected nucleic acid ligandable to bind and crosslink to a target molecule associated with adisease state is introduced into a patient in a number of ways known tothe art. For example, the photoSELEX ligand may be transiently orconstitutively expressed in the appropriate cells of a patient with thedisease. Alternatively, the photoSELEX ligand may be taken into apatient's cells as a double-stranded DNA which is transcribed in thecell in the presence of iodinated cytosine. Iodinated cytosine may beadministered to the patient, followed by irradiation with X-rays. ICincorporated into the nucleic acid ligand synthesized in the appropriatecells allows the ligand to crosslink and inactivate the target molecule.Further methods of introducing the photoSELEX ligand into a patientinclude liposome delivery of the halogenated ligand into the patient'scells.

[0121] Example 20 describes the production of modified nucleic acidligands that crosslink, with or without irradiation, to HIV-1 Revprotein. FIG. 15 shows the results of crosslinking to the bulk candidatemixture at various rounds of SELEX. rd1-round 1 pool RNA; rd7-round 7pool RNA; rd10-round 10 pool RNA; rd13-round 13 pool RNA; rd13/PK,photocrosslinked round 13 pool RNA proteinase K treated (35 μl of a 100μl reaction was incubated in 0.5% SDS, 50 μg/ml Proteinase K and 1 mMEDTA at 65 C. for one hour); rd13/no iU-round 13 pool RNA transcribedwith UTP (no iU). R-free RNA; XL-crosslinked nucleoprotein complex.

[0122]FIG. 16 shows the sequences sequenced after 13 rounds of SELEX(SEQ ID NOS:5-55). The sequences are aligned for maximum homology to the6a sequence (SEQ ID NO:5). Underlines represent potential base pairingas indicated by computer RNA folding algorithms. Dashed underlinesrepresent the 6a ligand “bubble” motif. Sequences flanked by underlinerepresent either loop or bulge regions. Dashes are placed to maximizealignment with 6a. * denotes that two isolates were obtained. +indicates laser independent crosslinking and − denotes the lack of laserindependent crosslinking to HIV-1 Rev. FIG. 17 (SEQ ID NOS:56-57) showsthe consensus for class 1 and class 2 ligands. FIGS. 18 and 19 show thesequence of Trunc2 (SEQ ID NO:58) and Trunc24 (SEQ ID NO:59) and thespecificity results. 500 nM protein, 20 μg tRNA, and approximately 1 nMof kinased trunc2 RNA were incubated for 10 min. at 37° C. andirradiated for 4 min. at 325 nm. t2-trunc2 RNA irradiated without addedprotein; t2Rev1/O′-trunc2 RNA, HIV-1 Rev protein and 0 min. ofirradiation; t2/Rev1/4′-trunc2 RNA, HIV-1 Rev protein, and 4 min. ofirradiation; t2/Rev1/4′/PK-trunc2 RNA, HIV-1 Rev protein, 4 min. ofirradiation, and proteinase K treated as in FIG. 1; t2/Rev2/4′-trunc2RNA, HIV-2 Rev protein, and 4 min. of irradiation; t2/Tat/4′-trunc2 RNA,HIV-1 Tat protein, and 4 min. of irradiation. R-free RNA; XL-crosslinkednucleoprotein complex. FIG. 20 shows the trunc24 photoindependentcrosslinking with HIV-1 Rev in the presence of human nuclear extract.

[0123] II. Solution SELEX

[0124] This embodiment of the present invention presents severalimproved methods for partitioning between oligonucleotides having highand low affinity for a target molecule. The method of the presentinvention has several advantages over prior art methods of partitioning:(1) it allows the isolation of nucleic acid ligands to the targetwithout also isolating nucleic acid ligands to the partitioning matrix;(2) it increases the speed and accuracy by which the oligonucleotidecandidate mixture is screened; and (3) the solution SELEX procedure canbe accomplished in a single test tube, thereby allowing the partitioningstep to be automated.

[0125] The materials and techniques required by the method of thepresent invention are commonly used in molecular biology laboratories.They include the polymerase chain reaction (PCR), RNA or DNAtranscription, second strand DNA synthesis, and nuclease digestion. Inpractice, the techniques are related to one another in a cyclic manneras illustrated in FIG. 21.

[0126] In the SELEX method, described by Tuerk and Gold (1990) Science249:1155 and illustrated in FIG. 21, a single-stranded nucleic acidcandidate mixture is generated by established procedures on a nucleicacid synthesizer, and is incubated with dNTP and Klenow fragment togenerate a population of double-stranded DNA templates. Thedouble-stranded DNA or the RNA transcribed from them are purified, andcontacted with a target molecule. RNA sequences with enhanced affinityto the target molecule form nucleic acid-target complexes. This isfollowed by partitioning of bound and unbound nucleic acids, andseparation of the target molecule from the bound nucleic acids. cDNA issynthesized from the enhanced affinity nucleic acids and double-strandedDNA generated by PCR amplification. The cycle is repeated until thecomplexity of the candidate mixture has decreased and its affinity aswell as specificity to the target has been maximized.

[0127] A novel feature of the solution SELEX method is the means bywhich the bound and free members of the nucleic acid candidate mixtureare partitioned. In one embodiment of the method of the presentinvention, generation of two physically distinct cDNA pools isaccomplished by use of primer extension inhibition. One cDNA extensionstep is added to the basic SELEX protocol between steps 2 and 3 above,which allows the generation of two physically distinct cDNA pools—onehaving high affinity for the target and one having low affinity for thetarget—which are easily distinguished and separated from each other.Primer extension inhibition analysis is a common technique for examiningsite-bound proteins complexed to nucleic acids (Hartz et al. (1988)Methods Enzymol. 164:419), and relies on the ability of high affinitycomplexes to inhibit cDNA synthesis. Examples of protein-nucleic acidinteractions studied by primer extension inhibition include ribosomebinding to the mRNA ribosome-binding site (Hartz et al. (1988) Meth.Enzym. 164:419) as well as binding of the unique E. coli translationfactor, SELB protein, to the mRNA selenocysteine insertion sequence(Baron et al. (1993) Proc. Natl. Acad. Sci. USA 90:4181).

[0128] In one embodiment of the solution SELEX scheme, the first cDNAextension is performed in the presence of chain terminating nucleotidetriphosphates. Under these conditions, oligonucleotides with lowaffinity for the target which form fast dissociating complexes with thetarget are converted into truncated cDNAs with a 3′-end terminated witha nonextendible nucleotide. The truncated cDNA chain is unable to annealto the PCR primers, and therefore, is non-amplifiable. In contrast,tight complexes formed between high affinity oligonucleotides and thetarget molecule, characterized by slow dissociation rates, inhibit cDNAextension. The chain terminators are not incorporated into the nascentcDNA chain synthesized from the high affinity oligonucleotide becausecDNA synthesis is blocked by the tightly bound target molecule. Fulllength cDNA from the high affinity complexes are obtained during asecond round of cDNA extension in which the target and chain terminatorshave been removed from the system. Thus, weak affinity complexes areconverted into truncated cDNA lacking the primer annealing site whiletight complexes are converted into fall length cDNA and are amplified byPCR (FIG. 22). The stringency of this method is easily modified byvarying the molar ratio of chain terminators and dNTPs or theconcentration of the polymerase, as primer extension inhibition issensitive to polymerase concentration (Ringquist et al. (1993)Biochemistry 32:10245). As used in the present disclosure, the term“stringency” refers to the amount of free RNA that will be convertedinto PCR product.

[0129] Therefore, one crucial feature of the invention is its ability topartition strong and weak affinity complexes into amplifiable andnon-amplifiable nucleic acid pools without requiring a partitioningmatrix. It is the unique properties of these cDNA pools that allowselective amplification of the high affinity ligand.

[0130] The target molecule can be a protein (either nucleic acid ornon-nucleic acid binding protein), nucleic acid, a small molecule or ametal ion. The solution SELEX method allows resolution of enantiomers aswell as the isolation of new catalytic nucleic acids.

[0131] Primer extension inhibition may be achieved with the use of anyof a number of nucleic acid polymerases, including DNA or RNApolymerases, reverse transcriptase, and Qβ-replicase.

[0132] The candidate mixture of nucleic acids includes any nucleic acidor nucleic acid derivative, from which a complementary strand can besynthesized.

[0133] Prior art partitioning included use of nitrocellulose or anaffinity column. One disadvantage of the prior art partitioning was thephenomenon of matrix binders in which nucleic acids that specificallybind the partitioning matrix are selected along with those thatspecifically bind the target. Thus, one advantage of the method of thepresent invention is that it overcomes unwanted selective pressureoriginating with use of a partitioning matrix by only using suchmatrixes after nucleic acids with high affinity for the target have beenpartitioned in solution and amplified. Moreover, the ability topartition strong and weak affinity complexes during cDNA synthesis,based on the ability of only the strongest complexes to inhibitextension by a polymerase, results in the selection of only the highestaffinity nucleic acid ligands. It is estimated that complexes withdissociation constants in the nanomolar or less range will efficientlyblock cDNA synthesis. The method of the present invention is expected topreferentially screen nucleic acid candidate mixtures for members thatbind the target at this limit.

[0134] The use of primer extension inhibition allows partitioning of theoligonucleotide candidate mixture into two pools—those oligonucleotideswith high target affinity (amplifiable) and those with low targetaffinity (non-amplifiable). As described above, chain terminators may beused to poison the first extension product, rendering the low affinitycDNAs non-amplifiable.

[0135] In another embodiment of the method of the present invention,restriction enzymes are used to selectively digest the cDNA generatedfrom the low affinity nucleic acids. A number of restriction enzymeshave been identified that cleave single-stranded DNA. These enzymescleave at specific sequences but with varying efficiencies. Partitioningof weak and strong affinity nucleic acids is accomplished by primerextension in the presence of the four dNTPs, followed by removal of thetarget and a second extension with modified nucleotides that areresistant to enzymatic cleavage. The cDNA pools can then be incubatedwith the appropriate restriction enzyme and the cDNA synthesized duringthe first extension cleaved to remove the primer annealing site andyield a non-amplifiable pool. Increased efficiency of cleavage isobtained using a hairpin at the restriction site (RS) to create alocalized double-stranded region (FIG. 24).

[0136] In another embodiment of method of the present invention, cDNAsequences corresponding to low affinity nucleic acids are renderedselectively degradable by incorporation of modified nucleotide into thefirst cDNA extension product such that the resulting cDNA ispreferentially degraded enzymatically or chemically.

[0137] In another embodiment of the method of the present invention, thefirst extension product can be removed from the system by an affinitymatrix. Alternatively, the matrix could be used to bind the secondextension product, e.g., the cDNAs corresponding to high affinitynucleic acids. This strategy relies on the incorporation of modifiednucleotides during cDNA synthesis. For instance, the first cDNAextension could be performed in the presence of modified nucleotides(e.g., biotinylated, iodinated, thiolabelled, or any other modifiednucleotide) that allow retention on an affinity matrix (FIG. 25). In analternate embodiment of the method of the present invention, a specialsequence can also be incorporated for annealing to an affinity matrix.Thus, first synthesis cDNAs can be retarded on commercially obtainablematrices and separated from second synthesis cDNA, synthesized in theabsence of the modified nucleotides and target.

[0138] In another embodiment of the invention, exonuclease hydrolysisinhibition is used to generate a pool of high affinity double-strandednucleic acid ligands.

[0139] In yet another embodiment of the invention, the solution SELEXmethod is used to isolate catalytic nucleic acids.

[0140] In another embodiment of the invention, solution SELEX is used topreferentially amplify single-stranded nucleic acids.

[0141] In a further embodiment of the invention, the solution SELEXmethod is automated.

[0142] Removal of the target to allow cDNA synthesis from the highaffinity nucleic acids can also be accomplished in a variety of ways.For instance, the target can be removed by organic extraction ordenatured by temperature, as well as by changes in the electrolytecontent of the solvent. In addition, the molecular repertoire of thecandidate mixture that can be used with the invention include any fromwhich a second complementary strand can be synthesized. Single-strandedDNA as well as RNA can be used, as can a variety of other modifiednucleotides and their derivatives.

[0143] The following non-limiting examples illustrate the method of thepresent invention. Example 21 describes the solution SELEX processwherein partitioning between high and low affinity nucleic acids isachieved by primer extension inhibition. Example 22 illustrates thesolution SELEX process wherein partitioning is achieved by restrictionenzyme digestion of low affinity RNA. Example 23 describes the solutionSELEX process wherein low affinity nucleic acids are separated from highaffinity nucleic acids by affinity chromatography. Example 24 describesthe isolation of high affinity double-stranded nucleic acid ligands withthe use of exonuclease inhibition. Example 25 describes the isolation ofcatalytic nucleic acids. Example 26 describes an automated solutionSELEX method.

[0144] The examples provided are non-limiting illustrations of methodsof utilizing the present invention. Other methods of using the inventionwill become apparent to those skilled in the art from the teachings ofthe present disclosure.

EXAMPLE 1

[0145] Synthesis of RNA Sequences RNA-1, RNA-2 RNA-3 and RNA-7 and R17Coat Protein

[0146] RNA-1 (SEQ ID NO:1), RNA-2 (SEQ ID NO:2), and RNA-3 (SEQ ID NO:3)shown in FIG. 6 and RNA-7 (SEQ ID NO:4) shown in FIG. 12 were preparedby in vitro transcription from synthetic DNA templates or plasmids usingmethodology described by Milligan and co-workers (Milligan et al. (1987)Nucleic Acids Res. 15:8783). Transcription reactions contained 40 mMtris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8.1 at 37°C.), 1 mM spermidine, 5 mM dithiothreitol (DTT), 50 μg/ml of bovineserum albumin (BSA), 0.1% (v/v) Triton X-100, 80 mg/ml of polyethyleneglycol (m_(r) 8000), and 0.1 mg/ml of T7 RNA polymerase. Largerquantities of RNA were prepared with 3-5 mM of each of the nucleotidetriphosphates (NTPs), 25 mM magnesium chloride, and 1 μM DNA template or0.1 μg/ml of plasmid. Body-labeled RNAs were prepared in 100 μMreactions with 1 mM each of the three NTPs, 0.25 mM of the equivalentradiolabelled NTP ([α-³²P] NTP, 5 μCi), 15 mM MgCl₂, and 0.1 mg/ml of T7RNA polymerase. Nucleotides, including 5-iodouridine triphosphate and5-bromouridine triphosphate, were obtained from Sigma Chemical Co., St.Louis, Mo. RNA fragments were purified by 20% denaturing polyacrylamidegel electrophoresis (PAGE). The desired fragment was eluted from thepolyacrylamide and ethanol-precipitated in the presence of 0.3 M sodiumacetate. R17 bacteriophage was propagated in Escherichia coli strainS26, and the coat protein was purified using the procedure described byCarey and coworkers (Carey et al. (1983) Biochemistry 22:4723).

EXAMPLE 2

[0147] Binding Constants for RNA-1 and RNA-2 to R17 Coat Protein

[0148] RNA-protein binding curves for hairpin variants RNA-1 (SEQ IDNO:1), RNA-2 (SEQ ID NO:2) and RNA-3 (SEQ I) NO:3) to the bacteriophageR17 coat protein are shown in FIG. 7. The association constants betweencoat protein and the RNA hairpin variants were determined with anitrocellulose filter retention assay described by Carey and co-workers(Carey et al. (1983) supra). A constant, low-concentration of³²P-labeled RNA was mixed with a series of coat protein concentrationsbetween 0.06 nM and 1 μM in 10 mM magnesium acetate, 80 mM KCl, 80 μg/mlBSA, and 100 mM Tris-HCl (pH 8.5 at 4° C.) (TMK buffer). These were thesame solution conditions used in the crosslinking experiments. Afterincubation at 4° C. for 45-60 min, the mixture was filtered through anitrocellulose filter and the amount of complex retained on the filterdetermined by liquid scintillation counting. For each experiment thedata points were fit to a non-cooperative binding curve and the K_(d)value shown in FIG. 7 was calculated.

EXAMPLE 3

[0149] Photocrosslinking of RNA-1 and RNA-2 to R17 Coat Protein at 308nm

[0150]³²P-Labeled RNA sequences RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ IDNO:2) (5 nM) and R17 coat protein (120 nM) were each incubated on ice in100 mM Tris-HCl (pH 8.5 at 4° C.), 80 mM KCl, 10 mM magnesium acetate,80 μg/ml of BSA for 15-25 min before irradiations. These are conditionsunder which the RNA is fully bound to the coat protein. The RNAs wereheated in water to 85° C. for 3 min and quick cooled on ice before useto ensure that the RNAs were in a hairpin conformation (Groebe andUhlenbeck (1988) Nucleic Acids Res. 16:11725). A Lambda Physik EMG-101excimer laser charged with 60 mbar of xenon, 80 mbar of 5% HCl in heliumand 2360 mbar of helium was used for 308 nm irradiations. The output ofthe XeCl laser was directed unfocused toward a 4 mm wide by 1 cm pathlength quartz cuvette containing the RNA-protein complex. The laser wasoperated in the range of 60 mJ/pulse at 10 Hz; however, only about 25%of the laser beam was incident upon the reaction cell. Photocrosslinkingyields of RNA-1 (SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) to the R17bacteriophage coat protein as a function of irradiation time are shownin FIG. 8. Crosslinked RNA was separated from uncrosslinked RNA by PAGE,and the yields were determined by autoradiography. Crosslinking of5-bromouracil-containing variant RNA-1 (SEQ ID NO:1) maximized at about40% because of competitive photodamage to the coat protein whichinhibits binding to the RNA (Gott et al. (1991) supra). Less photodamageto coat protein occurred with RNA 2 because of the shorter irradiationtime.

[0151] Crosslinking as a function of photons absorbed indicated that thequantum yield for crosslinking of BrU-RNA 1 is 0.014 and forcrosslinking of IU-RNA-2 (SEQ ID NO:2), 0.006 with irradiation at 308nm. In spite of the lower quantum yield, a higher crosslinking yield wasobtained with IU-RNA 2 because of the seven times higher absorptionprobability of the IU chromophore at 308 nm. BrU and IU absorb at 308 nmwith molar extinction coefficients of 385 and 2640 L/mol cm,respectively. Hence, a high level of photocrosslinking of the IU-RNA wasachieved prior to protein damage.

EXAMPLE 4

[0152] Identification of the Amino Acid Residue Involved in theCrosslink of RNA-1 to R17 Coat Protein

[0153] Large scale crosslinking of RNA-1 (SEQ ID NO:1) to R17 coatprotein. A 10 ml solution containing 300 nM 5′-end-labeled RNA and 500nM coat protein was incubated on ice in the presence of 100 mM Tris-HCl(pH 8.5 at 4° C.), 10 mM Mg(OAc)₂, 80 mM KCl, 80 mg bovine serum albumin(BSA), and 5 mM dithiothreitol (DTT) for 10-90 min. A Lambda PhysikEMG-101 excimer laser was used for monochromatic irradiation at 308 nm.The beam output was measured at 69±5 mJ/pulse at 10 Hz. Approximately50% of the beam was focused through a 7 mm-diameter circular beam maskinto a 1 cm path length quartz cuvette in a thermostated cell holder.The laser power was measured with a Scientech 360-001 disk calorimeterpower meter. The temperature was regulated at 4±2° C. with a Laude RC3circulating bath.

[0154] The 10 ml reaction mixtures were prepared just prior to theirradiations which were performed in 2 ml fractions. After 5 min ofirradiation the protein concentration was brought to 1 μM. The reactionmixture was then incubated for 3 min to allow exchange of photodamagedprotein for fresh protein in the nucleoprotein complex and irradiatedfor an additional 5 min. This step was repeated nine times to give 90 mlof irradiated sample. The crosslinking, analyzed by 20% denaturing PAGE,and quantitated on a Molecular Dynamics Phosphoimager, revealed 22%crosslinking.

[0155] The 90 ml sample contained 5.9 nmol of crosslinked RNA, 21 nmolof free RNA, 97 nmol of free coat protein, and 7.2 mg of BSA. The totalvolume was reduced to 20 ml and split equally between two 50 mlpolypropylene screw cap centrifuge tubes (Nalgene) and ethanolprecipitated overnight at −20° C. The RNA and proteins were spun down toa pellet at 13,000 rpm in a fixed angle J-20 rotor with an Beckman J2-21centrifuge. Each pellet was resuspended in 1 ml of 0.5 M urea, 50 mMTris-HCl pH 8.3, and 0.2% SDS for 48 h at 4° C. with shaking. Thefractions were combined, and the SDS was then removed by precipitationso as not to decrease the activity of trypsin. This was achieved using40 mM KCl, and the precipitate was removed by spinning through a 0.22 μmcellulose acetate spin filter. The trypsin conditions were optimizedusing 500 μl of the solution.

[0156] Proteolytic Digestion. The remaining 1.5 ml of crosslinked RNAsolution containing free RNA and protein was brought to 6 ml to contain1 M urea, 20 mM CaCl₂, and 6 mM DTT, and then 1.61 mg (1:5 w/w.)trypsin-TPCK (251 units/mg) was added. The reaction proceeded at 36° C.for 2 h at which time 1.61 mg more trypsin was added. At 4 h a 100 μLaliquot was removed and the reaction stopped by quick freezing. Thereaction was analyzed by 20% polyacrylamide 19:1 crosslinked, 7 M urea,90 mM Tris-borate/2 mM EDTA (TBE) gel electrophoresis (20% ureadenaturing PAGE).

[0157] Purification of the digested crosslinked RNA. The trypsinreaction mixture was brought to 10 ml to reduce the molar concentrationof salt, and run through a 240 μl DEAE ion exchange centrifuge column.The column was washed with 100 mM NaCl and spun dry in a bench topcentrifuge to remove free peptide. The column bound material containingthe RNA and crosslinked tryptic fragment was eluted from the column with1 ml of 600 mM NaCl and the column spun dry. An additional 200 μl of 600mM NaCl was spun through the column. The two fractions were pooled,ethanol precipitated and pelleted at 10,000 rpm for 35 min at 4° C. Thepellet was resuspended in 25 μl of 7 M urea-TBE buffer, 10 mM DTT, 0.1%bromophenol blue, 0.1% xylene cylanol, and heated to 85° C. for 4 minand purified by 20% denaturing PAGE. The gel ran for 3.5 h at 600 V. A 5min phosphoimage exposure was taken of the gel. The digested protein-RNAcrosslink was then electrolytically blotted from the gel onto a PVDFprotein sequencing membrane (0.2 micron) from Bio-RAD. The membrane wasair dried, coomassie stained for 1 min, destained for 2 min in 50% MeOH:50% H₂O, and rinsed twice with deionized H₂O. An autoradiogram was madeof the membrane to visualize the digested protein RNA crosslink whichwas excised from the membrane and submitted for Edman degradation. Theimmobilized peptide was sequenced by automated Edman degradation,performed on an Applied Biosystems 470A sequencer using manufacturer'smethods and protocols (Clive Slaughter, Howard Hughes Medical Institute,University of Texas, Southwestern). The Edman analysis indicated thatthe position of the crosslink was tyrosine-85 based upon the known aminoacid sequence Weber (1983) Biochemistry 6:3144).

EXAMPLE 5

[0158] Photocrosslinking of RNA-2 to R17 Coat Protein at 325 nm

[0159] In an experiment analogous to that described in Example 3,IU-substituted RNA-2 (SEQ ID NO:2) was photocrosslinked to R17 coatprotein with monochromatic emission at 325 nm from an Omnichrome HeCdlaser (model 3074-40M325). The power output of the HeCd laser was 37 mWand the total beam of diameter 3 mm was incident upon the sample. Toincrease excitation per unit time the beam was reflected back throughthe sample with a dielectric-coated concave mirror. Crosslinked RNA wasseparated from uncrosslinked RNA by PAGE, and the yields were determinedwith a PhosphoImager. The percent of the RNA crosslinked to the proteinas a function of irradiation time is shown in FIG. 9. High-yieldcrosslinking occurred without photodamage to the R17 coat protein. In aseparate experiment analogous irradiation of coat protein alone at 325nm with yet a higher dose resulted in protein which showed the samebinding constant to R17 coat protein. Irradiation at 325 nm ofBrU-containing RNA-1-R17 coat protein complex did not result incrosslinking because the BrU chromophore is transparent at 325 nm.

EXAMPLE 6

[0160] Photocrosslinking of RNA-1 and RNA-2 to R17 Coat Protein with aTransilluminator

[0161] In an experiment analogous to that described in Example 3, RNA-1(SEQ ID NO:1) and RNA-2 (SEQ ID NO:2) were photocrosslinked to the R17coat protein with broad-band emission in the range of 312 nm from aFisher Biotech Transilluminator (model FBTIV-816) filtered withpolystyrene. Crosslinked RNA was separated from uncrosslinked RNA byPAGE, and the yields were determined by autoradiography. Percent RNAscrosslinked to protein as a function of irradiation time is shown inFIG. 10.

EXAMPLE 7

[0162] Photoreaction of 5-iodouracil with N-Acetyltyrosine N-Ethyl Amide

[0163] N-acetyltyrosine N-ethylamide was prepared as described by Dietzand Koch (1987) supra. Iradiation of a pH 7, aqueous solution ofiodouracil and 10 mol equivalent excess of N-acetyltyrosine N-ethylamide at 308 nm with a XeCl excimer laser gave a photoadduct identicalto the photoadduct (structure 6) from irradiation of bromouracil andN-acetyltyrosine N-ethylamide (Dietz and Koch (1987) supra) as shown inFIG. 11. Product comparison was performed by C-18 reverse phase HPLC andby ¹H NMR spectroscopy. Although little is known about the mechanism ofphotocrosslinking of IU-substituted nucleic acids to associatedproteins, this result suggests that it is similar to that ofphotocrosslinking of BrU-substituted nucleic acids to associatedproteins.

EXAMPLE 8

[0164] Preparation of a cDNA from an RNA Photocrosslinked to a Protein

[0165] RNA-7 (SEQ ID NO:4) (FIG. 11) was prepared using methodology asreported in Example 1 using a plasmid instead of a DNA template. Thephotocrosslinking was performed as described in Example 3. A 4 mlreaction mixture consisting of 6.75 nM RNA and 120 nM R17 coat proteinwas irradiated, 2 ml at a time, at 308 nm with unfocused emission from aXeCl excimer laser. The laser produced 50 mJ/pulse and was operated at10 Hz. The reaction proceeded to near quantitative crosslinking, 85-90%,in 5 min of irradiation. After crosslinking, 1 ml of the total reactionmixture was removed; EDTA (80 mM), SDS (0.1%), and CaCl₂ (0.1 mM) wereadded; the free (unbound) RNA present was purified away; and the proteindigested with Proteinase K at 60° C. for 30 min. The RNA bound toresidual protein was ethanol precipitated to remove salts and spun to apellet. The pellet was washed three times with 70% ethanol to remove anyresidual salts. A reverse transcription reaction was employed to make acomplementary DNA copy of the RNA template. A 13-base promoter wasannealed to the RNA and the reverse transcription reaction was performedunder the standard conditions of the manufacturer, Gibco (Gaithersburg,Md.), and was stopped after 1 hr. The cDNA was body labelled with³²P-labelled deoxycytidine triphosphate. The RNA template was thenremoved by hydrolyzing with 0.2 M sodium hydroxide at 100° C. for 5 min.The formation of the cDNA was followed by PAGE. A hydrolysis ladder andmarkers were added to the gel to determine the length of the cDNA. ThecDNA co-migrated with the 44 nucleotide RNA template. If there had beena stop in the cDNA as a result of crosslinking modification, a shortenedproduct of 31 nucleotides would have been observed. A small amount of astop product was observed in the 22-25 nucleotide region of the gel, butthis may have resulted from the hairpin secondary structure which beginsat position 25 of the cDNA on the RNA template. No stop in the 31nucleotide region of the gel appeared; this established that the reversetranscriptase had read through the position of the crosslink. A diagramof the gel appears in FIG. 14.

EXAMPLE 9

[0166] Iodocytosine Photocrosslinking

[0167] 5-iodocytosine (IC) was incorporated in a hairpin RNA (RNA 8)that contained cytosine at the −5 position and bound the R17 coatprotein with high affinity. The IC-bearing RNA is designated RNA 9. RNA9 (5 nM) and R17 coat protein (120 nM) were incubated on ice in 100 mMTris-HCl (pH 8.5 at 4° C.)/80 mM KCl/10 mM magnesium acetate/ 80 μg/mlBSA for 15-25 min prior to irradiation. The RNA in water was heated to85° C. for 3 min and quick cooled on ice before use to ensure that itwould be in a conformation that bound the coat protein (Groebe andUhlenbeck (1988) supra). The complex was irradiated for 5 min at 4° C.,and the experiment was compared to control irradiations of RNA 2 and RNA8 coat protein complexes. Irradiation of RNA 8-coat protein complexresulted in no crosslinked product. Irradiation of RNA 9-coat proteincomplex resulted in the formation of a crosslink that formed in highyield (70-80%) similar to the yield of the control irradiation of RNA2-coat protein complex (80-90%) Crosslinking of RNA 9 is presumed tooccur through a similar mechanism as RNAs containing IU at position −5of the loop hairpin (FIGS. 6 and 12). This assumption is based on thespecificity of the crosslink since RNA 8 did not photocrosslink.

EXAMPLE 10

[0168] Incorporation of Halogenated Nucleotides into DNA Ligands

[0169] Photoreactive nucleotides may be incorporated into a DNA ligandcapable of crosslinking to a target molecule upon irradiation by themethods discussed above. 5-Bromodeoxyuracil (BrdU),8-bromo-2′-deoxyadenine, and 5-iodo-2′-deoxyuracil are examples of suchphotoreactive nucleotides.

EXAMPLE 11

[0170] PhotoSELEX

[0171] In one embodiment of the present invention, the photoSELEX methodis applied to completion in the selection of a nucleic acid ligand whichbinds and photocrosslinks to a target molecule.

[0172] A randomized set of nucleic acid oligonucleotides is synthesizedwhich contain photoreactive groups. The oligonucleotides of thecandidate mixture may be partially or fully saturated at each availableposition with a photoreactive group. The candidate mixture is contactedwith the target molecule and irradiated at the appropriate wavelength oflight. Oligonucleotides crosslinked to the target molecule are isolatedfrom the remaining oligonucleotides and the target molecule removed.cDNA copies of the isolated RNA sequences are made and amplified. Theseamplified cDNA sequences are transcribed into RNA sequences in thepresence of photoreactive groups, and the photoSELEX process repeated asnecessary.

EXAMPLE 12

[0173] Selection of Enhanced Photocrosslinking Ligands: SELEX Followedby PhotoSELEX

[0174] In one embodiment of the method of the present invention,selection of nucleic acid ligands through SELEX is followed by selectionthrough photoSELEX for ligands able to crosslink the target molecule.This protocol leads to ligands with high binding affinity for the targetmolecule that are also able to photocrosslink to the target.

[0175] Photoreactive nucleotides are incorporated into RNA by T7polymerase transcription with the reactive nucleotide triphosphate inplace of a specified triphosphate. For example, 5-bromouridinetriphosphate is substituted for uridine triphosphate or8-bromo-adenosine triphosphate is substituted for adenosinetriphosphate. A randomized set of RNA sequences containing photoreactivenucleotides are generated and the SELEX methodology applied. The initialSELEX rounds are used to eliminate intrinsically poor binders andenhance the pool of molecules that converge to form a pool of RNAs thatcontain the photoreactive group(s) and which bind to the targetmolecule. Aliquots from the initial SELEX rounds are irradiated and theenhancement of photocrosslinking followed via PAGE as the roundsproceed. As a slower migrating band representing crosslinked productsstarts to become evident, the pool of RNAs are introduced into rounds ofphotoSELEX. RNAs that have a photoreactive group adjacent to a reactiveamino acid residue in the nucleoprotein complexes form a crosslink andare selected and RNAs that do not have reactive nucleotides in proximityto reactive target residues are eliminated.

[0176] This protocol selectively applies photoSELEX selection topreviously identified ligands to a target molecule.

EXAMPLE 13

[0177] PhotoSELEX Followed by SELEX

[0178] In another embodiment of the method of the present invention, anRNA ligand able to photocrosslink a target molecule is preselectedthrough the photoSELEX methodology. Subsequently, SELEX is performed toselect a crosslinking oligonucleotide for ability to bind the targetmolecule.

EXAMPLE 14

[0179] Limited SELEX Followed by PhotoSELEX

[0180] In this embodiment of the present invention, nucleic acid ligandsare selected through the SELEX process for a limited number of selectionrounds. SELEX is not applied to completion as in Example 12. Rather, thecandidate mixture is partially selected for oligonucleotides havingrelatively enhanced affinity for the target molecule. The randomoligonucleotides of the candidate mixture contain photoreactive groupsand the initial SELEX selection is conducted in the absence ofirradiation. PhotoSELEX is then performed to select oligonucleotidesable to crosslink to the target molecule.

[0181] This protocol allows the selection of crosslinking ligands from apool of oligonucleotides with a somewhat enhanced capacity to bind thetarget molecule and may be useful in circumstances where selection tocompletion through SELEX does not yield crosslinking ligands.

EXAMPLE 15

[0182] Limited Directed PhotoSELEX

[0183] In one embodiment of the method of the present invention, inwhich nucleic acid ligands identified through SELEX are subjected tolimited randomization, followed by selection through photoSELEX.

[0184] The construction of the DNA template used to transcribe thepartially randomized RNA is based on the sequence of the initiallyselected ligand and contains at each position primarily the nucleotidethat is complementary to that position of the initial selected RNAsequence. However, each position is also partially randomized by usingsmall amounts of each of the other three nucleotides in the sequencer,which varies the original sequence at that position. A limited RNA poolis then transcribed from this set of DNA molecules with a photoreactivetriphosphate replacing a specific triphosphate in the reaction mix(i.e., BrU for U). The partially randomized set of RNA molecules whichcontains the photoreactive nucleotides is mixed with a quantity of thetarget protein. Bound RNAs that have a photoreactive group adjacent to areactive amino acid residue in the nucleoprotein complex form covalentcrosslinks upon irradiation. RNAs that bind and crosslink are selectedthrough several rounds of photoSELEX and separated away from RNAs thatbind but do not crosslink.

EXAMPLE 16

[0185] Methods for Modifying a Target Molecule

[0186] In another embodiment of the method of the present invention,photoSELEX is applied to develop a ligand capable of modifying a targetmolecule. Under these circumstances, incorporation of a photoreactivegroup onto or into a ligand selected by photo SELEX or SELEX may modifythe target in several ways such that the biological activity of thetarget molecule is modified. For example, the target molecule may beinactivated by photo crosslinked ligand. Mechanisms of inactivationinclude electron or hydrogen abstraction from the target molecule orradical addition to the target molecule that elicit a chemicalmodification. These different mechanisms may be achieved by changing themode of irradiation.

[0187] A ligand selected through photo SELEX used as a diagnostic for atarget molecule with ultraviolet (UV) light may also inactivate the sametarget in vivo if the source of irradiation is changed to X-rays orgamma rays. The resultant vinyl radical may work similarly to a hydroxylradical, that is, by abstraction of hydrogen atoms from the bindingdomain of the target molecule.

[0188] X-ray irradiation of the R17 coat protein bound to radiolabelledIU- or BrU-substituted RNA hairpin sequences may result in the formationof a crosslink. The BrU or I chromophore may also be excited to a higherenergy state by X-ray irradiation resulting in the formation of a Vinylradical (Mee (1987) in: Radiation Chemistry: Principles and Applications(Farhataziz and Rodgers, eds.), VCH Publishers, New York, pp. 477-499).The radical abstracts a hydrogen from the binding domain of the R17 coatprotein, thereby reducing or inhibiting its ability to bind the RNAligand. Inactivation is tested by X-ray irradiation of the R17 coatprotein in the presence and absence of substituted RNAs. The formationof crosslinked complexes is analyzed by PAGE. The effect of X-rayirradiation of RNA resulting in modification of binding by modificationof the protein domain is followed by nitrocellulose binding assay.

EXAMPLE 17

[0189] Diagnostic Use of PhotoSELEX to Identify Unique ProteinsAssociated with Specific Disease Processes

[0190] A goal of diagnostic procedures is to correlate the appearance ofunique proteins with specific disease processes. Some of thesecorrelations are obvious, e.g., after bacterial or viral infections, onecan detect antigens which are antigen specific or antibodies to suchantigens not found in the blood of uninfected subjects. Less obviouscorrelations include the appearance in serum of α-foeto protein which isdirectly correlated with the presence of the most common form oftesticular cancer.

[0191] The photoSELEX method may be applied to the discovery ofheretofore unknown correlations between biological proteins andimportant human diseases. In one embodiment of the present invention,serum is taken from a patient with a disease, RNA ligands to all theproteins in the serum are produced and adsorbed to normal sera. RNAligands to serum proteins may be identified through the SELEX method,with subsequent incorporation of photoreactive groups, or may beidentified through photo-SELEX, initially selected from a candidatemixture of oligonucleotides containing one or more photoreactive groups.RNA ligands left unbound are those which specifically bind only uniqueproteins in the serum from patients with that disease. For example, RNAligands are initially identified to a limited number of serum proteins(e.g., 11). The RNA ligands identified contain a modified NTP having areversible or photoreactive functional group capable of crosslinkingreversibly or non-reversibly with the target protein. Optionally, thepresence of a cross-linked ligand to every protein may be verified. TheRNA ligands are then removed and amplified. RNA is then transcribed fora second SELEX round. RNA is now bound to a large excess of 10 of theoriginal 11 proteins, leaving an RNA ligand specific for the unique(11th) protein. This RNA is then amplified. This is a subtractivetechnique.

[0192] In one embodiment of the diagnostic method of the presentinvention, the method described above is used to identify a ligand to anabnormal protein, for example, an α-foeto protein. Sera from patientswith important diseases is obtained and RNA ligands to all proteinspresent identified. The RNA ligands are adsorbed to normal sera, leavingan unbound ligand. The unbound ligand is both a potential diagnosticagent and a tool for identifying serum proteins specifically associatedwith a disease.

EXAMPLE 18

[0193] Method of Treating Disease by in vivo use of PhotocrosslinkingNucleic Acid Ligand

[0194] A nucleic acid ligand to a target molecule associated with adisease state is selected through the photoSELEX process (Example 11).The photoSELEX selected nucleic acid ligand may be introduced into apatient in a number of ways known to the art. For example, thenon-halogenated photoSELEX ligand is cloned into stem cells which aretransferred into a patient. The ligand may be transiently orconstitutively expressed in the patient's cells. IC administered to thepatient is incorporated into the oligonucleotide product of the clonedsequence. Upon irradiation, the ligand is able to crosslink to thetarget molecule. Irradiation may include visible, 325 nm, 308 nm, X-ray,ultraviolet, and infrared light.

[0195] Alternatively, the photoSELEX ligand may be taken into apatient's cells as a double-stranded DNA which is transcribed in thecell in the presence of iodinated cytosine. Further methods ofintroducing the photoSELEX ligand into a patient include liposomedelivery of the halogenated ligand into the patient's cells.

EXAMPLE 19

[0196] PhotoSELEX Ligands for use in in vitro Diagnostics in vivoImaging and Therapeutic Delivery

[0197] PhotoSELEX may be used to identify molecules specificallyassociated with a disease condition and/or abnormal cells such as tumorcells. PhotoSELEX-identified oligonucleotides may be produced that reactcovalently with such marker molecules.

[0198] In one embodiment of the present invention, the target forphotoSELEX is the abnormal serum or tumor cell (e.g., the targetmixture). A library candidate mixture of oligonucleotides is generatedcontaining photoreactive groups. Using one of the above-describedphotoSELEX protocols, oligonucleotides able to photocrosslink to theunique proteins in the abnormal serum or on the tumor cells areidentified. Oligonucleotides able to crosslink to a marker protein on atumor cell are useful as in vitro diagnostics or when coupled toenhancing agents for in vitro imaging. Further, oligonucleotides able tocrosslink to a marker protein on a tumor cell may be usedtherapeutically, for example, as a method for immune activation, as amethod of inactivation, or as a method of delivering specifictarget-active pharmaceutical compounds.

EXAMPLE 20

[0199] PhotoSELEX and HIV-1 Rev

[0200] At each position of the template deoxy-oligonucleotide synthesis,the nucleotide reagent ratio was 62.5:12.5:12.5:12.5. The nucleotideadded in greater amount at each position corresponds to the nucleotidefound in the 6a sequence (SEQ ID NO:5) at the same position.

[0201] Cloning and Sequencing procedure: RNA's isolated from each roundwere reverse transcribed to produce cDNA and PCR amplified producing a111 bp fragment with unique BamHI and HindIII restriction sites at theends. The phenol/CHCl₃ treated fragment and a pUC18 vector were digestedtogether overnight with BamHI and HindIII at 37° C., phenol/CHCl₃treated and precipitated. The digested vector and PCR product wasligated at room temperature for 4 hours with T4 DNA ligase andtransformed to competent DH5α-F′cells which were then grown onampicillin-containing LB plates. Individual colonies were grownovernight in LB-ampicillin media and plasmid was prepared using Wizard(Promega) plasmid preparation kit. Sequencing was performed utilizing aSequenase (USB) kit.

[0202] Conditions for nitrocellulose filter binding selections: Allrounds utilized approximately 20 nM RNA. Round 1 and 2: 6 nM Rev. Round3: 3 nM Rev. Round 8: 1 nM Rev. Round 9-10: 3 nM Rev. Binding reactionvolumes ranged from 5 mls to 1 ml.

[0203] Conditions for crosslinking selections: Approximately 50-100 nMof folded pool RNA was added to 0.2 (Rounds 4-6) or 0.5 (Round 7) μMRev, 1 μM BSA in 1×BB (50 mM TrisAc pH 7.7, 200 mM KOAc, 10 mM DTT) onice and incubated 5 minutes at 37° C. The samples were then irradiatedat 37° C., for 3 minutes at 308 nm by a XeCl excimer laser (round 4), 30minutes at 325 nm by a HeCd laser (round 5), 10 minutes at 325 nm,(round 6), or 1 minute at 325 nm, (round 7). Approximately one-half ofthe sample was heated in 50% formamide, 40 μg tRNA at 90° C. for 4minutes and separated by electrophoresis in an 8 percentpolyacrylamide/8 M urea gel.

[0204] The following procedure was utilized to elute crosslinked RNAsfrom acrylamide gels with approximately 80% recovery: The nucleoproteincontaining gel slice was crushed to a homogenous slurry in 1×PK buffer(100 mM Tris-Cl pH 7.7, 50 mM NaCl and 10 mM EDTA). Proteinase K wasadded to 1 mg/ml concentration and incubated at 42° C. for 30 minutes.Fifteen minute incubations at 42° C. with increasing urea concentrationsof approximately 0.7 M, 1.9 M, and 3.3M were performed. The resultingsolution was passed through DMCS treated glass wool and 0.2 μm celluloseacetate filter. The filtered solution was extracted twice withphenol/CHCl₃ and then precipitated with a 1:1 volume mixture ofEtOH:isopropanol.

[0205] The crosslinked band from each round was placed in scintillationfluid and counted in a Beckman LS-133 Liquid Scintillation System. Thepercent crosslinked=cpms of crosslinked product from RNA+Rev after 4minutes irradiation at 325 nm minus cpms in crosslinked region for RNAonly irradiated divided by total cpms. The fold increase in crosslinkingis % R13 crosslinked divided by % D37 crosslinked.

[0206] Simultaneous selection for affinity and crosslinking usingcompetitor tRNA was performed as follows. 10 μM yeast tRNA was added to0.5 μM Rev, 1 μM BSA in 1×BB (50 mM TrisAc (pH=7.5), 200 mM KOAc, 10 mMDTT) and incubated 10 minutes on ice. 200,000 cpms (approximately 50-100nM final concentration RNA) was added and incubated an additional 15 to60 minutes on ice followed by 5 minutes at 37° C. The samples were thenirradiated 4 minutes at 325 nm by a HeCd laser at 37° C. Approximatelyone-third of the sample was heated in 50% formamide, 40 μg tRNA at 90°C. for 4 minutes and separated by electrophoresis in an 8 percentpolyacrylamide-8M urea gel.

[0207] The LI crosslinking RNA ligands form additional crosslinkedproduct with a 4 minute 325 nm laser irradiation.

[0208] The template oligos used to produce the truncated RNA's are:PTS-1; 5′-TAATACGACTCACTATA-3′, (SEQ ID NO:60) DNA-2;5′-GAGTGGAAACACACGTGGTGTTT-CATACACCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO:61),and DNA-24; 5′-AGGGTTAACAGGTGTGCCTGTTAATCCCCTATAGT-GAGTCGTATTA-3′ (SEQID NO:62).

[0209] PTS-1 was annealed with DNA-2 or DNA-24 to produce a template forT7 transcription.

[0210] To calculate the number of changes for individual moleculescompared to 6a (SEQ ID NO:5), each was aligned to 6a for maximumsimilarity. Gaps are calculated as one change and truncated moleculeswere counted as unchanged. To calculate the average probability offinding molecules within each class; the average number of specific (s)and non-specific (ns) changes and unchanged (u) were calculated and usedin the equation: (P)=(0.125)^(s)(0.375)^(ns)(0.625)^(u). Class Ia(P)=9×10⁻¹⁵; Ib (P)=3×10⁻¹⁵; Ic (P)=7×10⁻¹³; Id (P)=3×10⁻¹⁵; ClassII(P)=2×10⁻¹⁴. Since the starting population consists of 10¹⁴ molecules,sequences with (P)<10⁻¹⁴ will not be represented. (s) are those changesrequired to produce the uppercase, consensus nucleotides and (ns) areadditional changes.

[0211] Trunc24 (SEQ ID NO:59) photo-independent crosslinking with HIV-1Rev in the presence of human nuclear extracts was determined as follows:Trunc24 RNA, nuclear extracts, and Rev protein were combined andincubated on ice for 10 min. Samples were mixed 1:1 with 8 M urealoading buffer and placed on a 7 M urea, 8% polyacrylamide gel foranalysis, XL indicates the nucleoprotein complex, RNA indicates freetrunc24 RNA.

EXAMPLE 21

[0212] Primer Extension Inhibition Solution SELEX

[0213] Primer extension inhibition relies on the ability of a tightlybound target molecule to inhibit cDNA synthesis of high affinityoligonucleotides and results in formation of an amplifiable cDNA poolcorresponding to high affinity oligonucleotides and a non-amplifiablecDNA pool corresponding to low affinity oligonucleotides. Thus, the PCRstep of solution SELEX acts as a partitioning screen between two cDNApools. General protocols for nucleic acid synthesis, primer extensioninhibition and PCR are herein provided. Further, N-acryloylamino phenylmercuric gel electrophoretic conditions for separation of selectednucleic acid ligands is described. The methods of cloning and sequencingnucleic acid ligands is as described by Tuerk and Gold (1990) supra.

[0214] RNA Synthesis. The RNA candidate mixture was generated byincubating RNA polymerase and DNA templates. The reaction conditions are8% polyethylene glycol 8000, 5 mM dithiothreitol, 40 mM Tris-HCl (pH8.0), 12 mM MgCl₂, 1 mM spermidine, 0.002% Triton X-100, 2 mM nucleotidetriphosphates, and 1 unit/μl RNA polymerase. Reactions are incubated at37° C. for 2 hours.

[0215] The transcription protocol may be used to generate RNAs withmodified nucleotides. The transcription reaction may either be primedwith a nucleotide triphosphate derivative (to generate a modified 5′end), modified nucleotides may be randomly incorporated into the nascentRNA chain, or oligonucleotides or their derivatives ligated onto the 5′or 3′ ends of the RNA product.

[0216] Primer Extension Inhibition. Primer extension inhibition isperformed as described by Hartz et al. (1988) supra. Briefly, anoligonucleotide primer is annealed to the 3′ end of the oligonucleotidesof the candidate mixture by incubating them with a 2-fold molar excessof primer at 65° C. for 3 min in distilled water. The annealing reactionis cooled on ice, followed by the addition of 1/10 volume of10×concentrated extension buffer (e.g., 10 mM Tris-HCl (pH 7.4), 60 mMNH₄Cl, 10 mM Mg-acetate, 6 mM β-mercaptoethanol, and 0.4 mM nucleotidetriphosphates). Primer extension is initiated by addition of polymeraseand incubation at any of a variety of temperatures ranging between 0-80°C., and for times ranging from a few seconds to several hours. In oneembodiment of the method of the present invention, primer extension isfirst conducted in the presence of chain terminating nucleotidetriphosphates such that low-affinity nucleic acids preferentiallyincorporate these chain terminators. A second primer extension is thenconducted after removing the target from high affinity nucleic acids andremoving the chain terminating nucleotides triphosphates.

[0217] Polymerase Chain Reaction. The polymerase chain reaction (PCR) isaccomplished by incubating an oligonucleotide template, either single-or double-stranded, with 1 unit/μl thermal stable polymerase in buffer(50 mM KCl, 10 mM Tris-HCI (pH 8.6), 2.5 mM MgCl₂, 1.7 mg/ml BSA, 1 mMdeoxynucleotide triphosphates, and 1 μM primers). Standard thermalcycles are 95° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 1 min.,repeated as necessary. One modification of the PCR protocol generatessingle-strand DNA by incubating either single- or double-strandedtemplate with a single, elongated primer oligonucleotides and results inan elongated product. PCR preferentially amplifies the oligonucleotidesrendered amplifiable in the primer extension steps described above.

[0218] (N-Acryloylamino)phenyl mercuric gel electrophoresis.Polyacrylamide gel electrophoresis using N-acryloylamine phenyl mercury(APM) was performed as described by Igloi (1988) Biochemistry 27:3842.APM was synthesized by mixing 8 ml of acetonitrile to 0.35 g of(p-aminophenyl)mercuric acetate at 0° C., followed by 2 ml of 1.2 MNaHCO₃. A total of 0.2 ml of acryloyl chloride was then added withvigorous stirring and the reaction incubated overnight at 4° C. Thesolid phase was collected by centrifugation and washed with water,dissolved by warming to 50° C. in 8.5 ml of dioxane, followed byfiltration to remove undissolved contaminants. APM crystals were formedupon standing at room temperature and the solid was washed again withwater and dried under vacuum. APM was stored at 4° C. APM-polyacrylamidegels were prepared by addition of a appropriate aliquot of a 1 mg/mlsolution of APM in formamide to a solution containing a given amount ofacrylamide, bis(acrylamide), an urea in 0.1 M Tris-borate/EDTA (pH 8.3).Polymerization was initiated by addition of 0.5 ml of 1% ammoniumpersulfate and 7 μl of TEMED per 10 ml of gel solution.

EXAMPLE 22

[0219] Enzymatic or Chemical Degradation Solution SELEX

[0220] Enzymes or chemicals may be used to selectively degrade the poolof cDNA corresponding to low-affinity oligonucleotides. In oneembodiment of the present invention, restriction enzymes are used toselectively degrade the cDNA pool corresponding to low-affinityoligonucleotides. A number of restriction enzymes have been identifiedthat cleave single-stranded DNA. These enzymes cleave at specificsequences but with varying efficiencies.

[0221] Restriction enzyme digestion may be performed with a variety ofsequence specific restriction endonucleases. Endonucleases that cleavesingle-stranded DNA include DdeI, HaeIII, HgaI, HinfI, HinPI, MnII,PstI, and RsaI. These enzymes are used under standard conditions knownto those skilled in the field of molecular biology. Double-strandednucleic acids may also be cleaved using the proper combination ofnucleic acid restriction sequences and site specific restrictionnucleases.

[0222] The basic solution SELEX procedure is followed as described inthe SELEX Patent Applications. The first cDNA extension is performed inthe presence of four dNTPS, followed by removal of the target. Thesecond cDNA extension is performed with modified nucleotides that areresistant to enzymatic cleavage by restriction endonucleases. Themixture of cDNA extension products is incubated with the appropriaterestriction enzyme. The product of the first cDNA extension from freenucleic acid is cleaved to remove the primer annealing site, renderingthis cDNA pool non-amplifiable by PCR. The efficiency of cleavage byrestriction endonucleases may be improved using a hairpin at therestriction site (RS) to create a localized double-stranded region, asshown in FIG. 24.

[0223] Alternatively, the first cDNA extension product is renderedselectively degradable by other classes of enzymes by incorporation ofmodified nucleotides. For example, cDNA corresponding to low affinityligands may be synthesized with nucleotides sensitive to uracil DNAglycosylase, while cDNA corresponding to high affinity ligands mayincorporate resistant nucleotides.

[0224] Chemical degradation of cDNA corresponding to low affinityligands can be accomplished by incorporation of 7-methylguanosine,5-bromouracil, or 5-iodouracil as described using piperidine orphotodegradation (Sasse-Dwight and Gralla (1991) Methods Enzymol.208:146; Aiken and Gumport (1991) Methods Enzymol. 208:433; Hockensmithet al. (1991) Methods Enzymol. 208:211).

EXAMPLE 23

[0225] Solution SELEX Followed by Affinity Chromatography

[0226] Selective removal of either the first or second cDNA extensionproducts may be achieved through affinity chromatography. Removal of thefirst cDNA extension product preferentially removes the cDNA poolcorresponding to free or low-affinity nucleic acids. Removal of thesecond cDNA extension product preferentially retains cDNA correspondingto the high-affinity ligand. This strategy relies on the incorporationof modified nucleotides during cDNA synthesis.

[0227] Selective Removal of First Extension Product. Following the basicsolution SELEX protocol, the first cDNA extension is performed in thepresence of modified nucleotides (e.g., biotinylated, iodinated,thiolabelled, or any other modified nucleotide) that allow retention ofthe first cDNA pool on an affinity matrix (FIG. 25). The target is thenremoved and the second cDNA extension performed in the presence ofnon-modified nucleotides. The cDNAs that have incorporated the modifiednucleotides may be removed by affinity chromatography using a columncontaining the corresponding affinity ligand. The cDNA poolcorresponding to nucleic acids with high affinity for the target remainand are then amplified by PCR.

[0228] Selective Removal of the Second Extension Product. Following thebasic protocol, the first cDNA extension is performed in the presence offour dNTPs, and the second cDNA extension is performed in the presenceof modified nucleotides (e.g., biotinylated, iodinated, thiolabelled, orany other modified nucleotide) that allow retention of the second cDNApool on an affinity matrix as described above.

[0229] Incorporation of Specific Sequences for Annealing to An AffinityMatrix. In an alternate embodiment of the method of the presentinvention, a special sequence can also be selectively incorporated forannealing to an affinity matrix. Thus, either first or second synthesiscDNAs can be retarded and purified on commercially obtainable matricesas desired.

EXAMPLE 24

[0230] Exonuclease Inhibition Solution SELEX

[0231] Exonuclease inhibition may be used to isolate double-strandedligands. Double-stranded nucleic acid ligands tightly bound to thetarget molecule will inhibit exonuclease hydrolysis at the 3′ edge ofthe binding site. This results in a population of nucleic acid moleculesresistant to hydrolysis that also contain a long single-stranded 5′overhang and a central base paired region (see FIG. 26). This nucleicacid molecule is a substrate for any polymerase, and incubation withpolymerase will generate the double-stranded starting material. Thismolecule is amplified by PCR. Members of the nucleic acid candidatemixture that are not tightly bound to the target molecule are digestedduring the initial exonuclease step.

[0232] 3′→5′ hydrolysis of double-stranded nucleic acid is accomplishedby incubation with any double-stranded specific 3′→5′ exonuclease.Exonuclease III specifically hydrolyzes double-stranded DNA 3′→5′ and isactive in a variety of buffers, including 50 mM Tris-HCl (pH 8.0), 5 mMMgCl₂, 10 mM β-mercaptoethanol at 37° C.

EXAMPLE 25

[0233] Solution SELEX Method for Isolating Catalytic Nucleic Acids

[0234] Solution SELEX may be used to isolate catalytic nucleic acidsequences. This embodiment of the invention takes advantage of a linearto circular transformation to sort non-catalytic nucleic acids fromcatalytic nucleic acids.

[0235] As shown in FIG. 27, the PCR step may be exploited to screen thenucleic acid candidate mixture for catalytic members. Catalytic nucleicacids that either self-circularize, or alter their 5′ or 3′ ends toallow circularization with ligase, will amplify during PCR. The figureillustrates circle formation by catalytic members of the candidatemixture; the non-catalytic oligonucleotide members of the candidatemixture will remain linear. After circularization, the candidate mixtureis incubated with a primer that anneals to the extreme 5′ end. In thisembodiment of the invention, only the circular oligonucleotide memberswill generate cDNA and be amplified during the PCR step.

[0236] This strategy isolates nucleic acids that either directlycatalyze self-circularization or that modify their own ends so that theamplifiable form may be generated by incubation with ligase. As shown inFIG. 27, the unusual interaction of the cDNA primer with the 5′ end ofthe oligonucleotides of the candidate mixture permits amplification ofonly the circular molecules. In a further embodiment of the method ofthe present invention, this strategy is modified to allow isolation ofcatalytic nucleic acids that catalyze novel reactions.

EXAMPLE 26

[0237] Automation of Solution SELEX

[0238] The automated solution SELEX protocol represents a modificationof the technology used in the automated DNA synthesizer. The nucleicacid candidate mixture is attached to a solid support by either thebiotin/avidin interaction or a variety of covalent chromatographictechniques (e.g., the condensation of modified nucleotides ontomaleimide or citraconic anhydride supports). The bound nucleic acidcandidate mixture provides a good substrate for targeting binding, andthe column allows use of a single reaction vessel for the SELEXprocedure. Primer extension inhibition is used to physically sort lowand high affinity ligands. Low affinity nucleic acids may be degraded byincorporation of modified nucleotides during the first cDNA extensionstep that renders the cDNA degradable as described in Example 22, whilehigh affinity ligands are copied into non-degradable cDNA and amplifiedby PCR. For additional rounds of solution SELEX, the PCR generatedcandidate mixture is purified or is transcribed into RNA and reattachedto a second solid support, in the same or a new reaction vessel asdesired. The process is repeated as necessary.

1 64 19 base pairs nucleic acid single linear U at position 13 is 5-bromouracil 1 GGGAGCGAGC AAUAGCCGC 19 19 base pairs nucleic acid singlelinear U at position 13 is 5- iodouracil 2 GGGAGCGAGC AAUAGCCGC 19 19base pairs nucleic acid single linear U at position 13 has hydrogenmolecule attached 3 GGGAGCGAGC AAUAGCCGC 19 44 base pairs nucleic acidsingle linear all U are 5-iodouracil 4 GAACAUGAGG AUUACCCAUG AAUUCGAGCUCGCCCGGGCU CUAG 44 37 base pairs nucleic acid single linear 5 GGGUGCAUUGAGAAACACGU UUGUGGACUC UGUAUCU 37 36 base pairs nucleic acid singlelinear 6 AGGUACGAUU AACAGACGAC UGUUAACGGC CUACCU 36 37 base pairsnucleic acid single linear 7 UAACGGCUUA ACAAGCACCA UUGUUAACCU AGUGCCU 3737 base pairs nucleic acid single linear 8 GAGUGGCUUA ACAAGCACCAUUGUUAACCU AGUACCU 37 36 base pairs nucleic acid single linear 9GUGCAGAUUA ACAACAACGU UGUUAACUCC UCCUCU 36 37 base pairs nucleic acidsingle linear 10 CUGUGGAUUA ACAGGCACAC CUGUUAACCG UGUACCU 37 37 basepairs nucleic acid single linear 11 CUGUGGAUUA ACAGGCACAC CUGUUAACCGUGUACCC 37 36 base pairs nucleic acid single linear 12 AGACGAUUAACAUCCACGGA UGUUAACGCG CUAGAA 36 37 base pairs nucleic acid single linear13 AAGACGAUUA ACAAACACGU UUGUUAACGC AACACCU 37 36 base pairs nucleicacid single linear 14 GAUUGGAUUA ACAGGCACCC CUGUUAACCU ACCACU 36 37 basepairs nucleic acid single linear 15 AGGAGGAUUA ACAACAAAGG UUGUUAACCCCGUACCA 37 34 base pairs nucleic acid single linear 16 UGAAGGAUUAACAACUAAUG UUGUUAACCA UGUA 34 37 base pairs nucleic acid single linear17 UUGAGGAUUA ACAGGCACAC CUGCUAACCG UGUACCC 37 37 base pairs nucleicacid single linear 18 AUGUGGCUUA ACAAGUACGC UUGUUAACCC AAAAACG 37 35base pairs nucleic acid single linear 19 AGGACGAUGA ACAAACACGUUUGUUCACGC CAUGC 35 38 base pairs nucleic acid single linear 20GACUGGCUUA ACAAACAUGU UUUGUUAACC GUGUACCA 38 37 base pairs nucleic acidsingle linear 21 CGGCGGAUUA ACACGACACA CUCGUGUUAA CCAUAUC 37 37 basepairs nucleic acid single linear 22 GCAUCAGAUG AACAGCACGU CUGUUCACUAUGCACCC 37 37 base pairs nucleic acid single linear 23 GCAUCAGAUGAACAGCACGU CUGUUCACUA UGCACCU 37 37 base pairs nucleic acid singlelinear 24 GCAUCAGAUG GACAGCACGU CUGUUCACUA UGCACCU 37 37 base pairsnucleic acid single linear 25 CAGUGUAUGA AACACCACGU GUGUUUCCAC UGUACCU37 35 base pairs nucleic acid single linear 26 CAGUGUAUGA AACAACACGUUUGUUUCCAC UGCCU 35 35 base pairs nucleic acid single linear 27GAGUGUAUGA AACAACACGU UUGUUUCCAC UCCCU 35 35 base pairs nucleic acidsingle linear 28 GAGUGUAUGA AACAACACGU UUGUUUCCAC UGUCU 35 35 base pairsnucleic acid single linear 29 GAUUGUAUGA AACAACGUGU UUGUUUCCAC UCCCU 3535 base pairs nucleic acid single linear 30 GAAUGUAUGA AACAACACGUUUGUUUCCAC UGCCU 35 37 base pairs nucleic acid single linear 31GAUUGGACUU AACAGACACC CCUGUUAACC UACCACU 37 34 base pairs nucleic acidsingle linear 32 UGCGACAGUU AGAAACACGA UUGUUUACUG UAUG 34 36 base pairsnucleic acid single linear 33 UACAGGCUUA AGAAACACGU UUGUUAACCA ACCCCU 3636 base pairs nucleic acid single linear 34 UCGAGCAGUG UGAAACACGAUUGUGUUUCC UGCUCA 36 36 base pairs nucleic acid single linear 35UGAUGCCUAG AGAAACACAU UAGUGUUUCC CUCUGU 36 37 base pairs nucleic acidsingle linear 36 ACGUGCCUCU AGAAACACAU CUGAUGUUUC CCUCUCA 37 37 basepairs nucleic acid single linear 37 ACCCGCCUCG UGAAACACGC UUGAUGUUUCCCUCUCA 37 34 base pairs nucleic acid single linear 38 CGGUGACGUAUGAAACACGU UCGUUGAUUU CCGU 34 30 base pairs nucleic acid single linear39 GCUUGCGAAA CACGUUUGAC GUGUUUCCCU 30 33 base pairs nucleic acid singlelinear 40 GCACCCUAGA AACGCGUUAG UAGACGUUUC CCU 33 37 base pairs nucleicacid single linear 41 AGGAACCUAG AAACACACAG UGUUUCCCUC UGCCCAC 37 37base pairs nucleic acid single linear 42 GCCUGCAUGG AUUAACACGUAUGUGUUAAC CGACUCC 37 37 base pairs nucleic acid single linear 43UGAAACACUG AGAAACACGU GUUUCCCCUU GUGUGAU 37 36 base pairs nucleic acidsingle linear 44 AGGAACCUCA AGCCGCCCCU AGAACACUCG GCACCU 36 37 basepairs nucleic acid single linear 45 AGGAACCUCA AGAAAGCCCC UGAAACACUCGAAGCCU 37 37 base pairs nucleic acid single linear 46 AGGAACCUCAAGAAACCCCC UGAAACACUC AUUACCG 37 37 base pairs nucleic acid singlelinear 47 AGGAACCUCA AGAAAUCCGA ACGACAACCC UACACCU 37 36 base pairsnucleic acid single linear 48 AGGAACCUCA AGAAACCCCG CCACGGACCC CAACCA 3637 base pairs nucleic acid single linear 49 GGGAACCUCA AUAAUCACGCACGCAUACUC GGCAUCU 37 34 base pairs nucleic acid single linear 50GGGAACCUCA AGAGACCCGA CAGGAUACUC GGAC 34 37 base pairs nucleic acidsingle linear 51 AAGUGGAACC UCAAUCCCGU AAGAAGAUCC UGUACCU 37 37 basepairs nucleic acid single linear 52 AUGUGCAUAG AGAUGUACAU AUGAAACCUCAGUAGAG 37 37 base pairs nucleic acid single linear 53 UCAUGCAUAGGCAUAGGCAG AUGGAACCUC AGUAGCC 37 37 base pairs nucleic acid singlelinear 54 AUGUGCAACA AGGCGCACGG AUAAGGAACC UCGAAGU 37 37 base pairsnucleic acid single linear 55 GAGUACAGCA CGCAACACGU ACGGGGAACC UCAAAGU37 20 base pairs nucleic acid single linear N at positions 1 and 20indicates 1-2 complementary base pairs N at position 3 indicates 1 or 3nucleotides N at positions 10 and 12 indicates 1-4 complementary basepairs N at position 11 indicates 4 or 5 nucleotides U is iodouracil 56NGNKDAACAN NNUGUUHMCN 20 23 base pairs nucleic acid single linear U isiodouracil 57 GGAACCUCAA UUGAUGGCCU UCC 23 32 base pairs nucleic acidsingle linear U is iodouracil 58 GGGUGUAUGA AACACCACGU GUGUUUCCAC UC 3229 base pairs nucleic acid single linear 59 GGGGAUUAAC AGGCACACCUGUUAACCCU 29 17 base pairs nucleic acid single linear 60 TAATACGACTCACTATA 17 49 base pairs nucleic acid single linear 61 GAGTGGAAACACACGTGGTG TTTCATACAC CCTATAGTGA GTCGTATTA 49 46 base pairs nucleic acidsingle linear 62 AGGGTTAACA GGTGTGCCTG TTAATCCCCT ATAGTGAGTC GTATTA 4613 base pairs nucleic acid single linear 63 CTAGAGCCCG GGC 13 44 basepairs nucleic acid single linear 64 CTAGAGCCCG GGCGAGCTCG AATTCATGGGTAATCCTCAT GTTC 44

1. A nucleic acid ligand that photocrosslinks to a target molecule thatis associated with a disease state, wherein said nucleic acid ligand iscomprised of a non-naturally occurring nucleic acid having a specificbinding affinity for a molecule, wherein said molecule is not a nucleicacid binding molecule, and wherein said nucleic acid ligand is not anucleic acid having the known physiological function of being bound bythe molecule, obtained by the process of: a) identifying a nucleic acidligand that photocrosslinks to a target molecule that is associated witha disease state from a candidate mixture of nucleic acids, wherein eachmember of said candidate mixture contains a photoreactive group, saidmethod comprising: i) contacting said candidate mixture of nucleic acidswith a first biological substance which contains a target molecule thatis associated with said disease state, wherein nucleic acids having anincreased affinity to a molecule of said first biological substancerelative to the candidate mixture form nucleic acid-molecule complexeswith the molecule; ii) irradiating said complexes, wherein said nucleicacid and molecule photocrosslink; iii) partitioning the photocrosslinkednucleic acid-molecule complexes from the remainder of the candidatemixture; and iv) identifying nucleic acid ligands that photocrosslink tosaid molecule; b) contacting a second biological substance which doesnot contain said target molecule that is associated with said diseasestate with said nucleic acid ligand identified in step iv), wherein thenucleic acids with affinity to the molecule that is not associated withthe disease state in the second biological substance is removed; and c)amplifying the remaining nucleic acids with specific affinity to saidmolecule that is associated with said disease state to yield a mixtureof nucleic acids enriched for nucleic acids with relatively higheraffinity and specificity for binding to said target molecule that isassociated with said disease state, whereby a nucleic acid ligand to atarget molecule that is associated with a disease state in a biologicalsubstance is identified.
 2. The nucleic acid ligand of claim 2 furthercomprising one or more of the photoreactive groups selected from thegroup consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine,5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine,5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine,8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine,8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine,8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine.
 3. A nucleic acid ligand thatphotocrosslinks to a target molecule that is associated with a diseasestate, wherein said nucleic acid ligand is comprised of a non-naturallyoccurring nucleic acid having a specific binding affinity for amolecule, wherein said molecule is not a nucleic acid binding molecule,and wherein said nucleic acid ligand is not a nucleic acid having theknown physiological function of being bound by the molecule, obtained bythe process of: a) identifying a nucleic acid ligand thatphotocrosslinks to a target molecule that is associated with a diseasestate from a candidate mixture of nucleic acids, said method comprising:i) contacting said candidate mixture of nucleic acids with a firstbiological substance which contains a target molecule that is associatedwith said disease state, wherein nucleic acids having an increasedaffinity to a molecule of said first biological substance relative tothe candidate mixture form nucleic acid-molecule complexes with themolecule; ii) partitioning the complexed increased affinity nucleicacids from the remainder of the candidate mixture; iii) amplifying theincreased affinity nucleic acids to yield a ligand-enriched mixture ofnucleic acids, iv) incorporating photoreactive groups into saidamplified increased affinity nucleic acids; v) irradiating saidincreased affinity nucleic acids, wherein said nucleic acid-moleculecomplexes photocrosslink; vi) partitioning the photocrosslinked nucleicacid-molecule complexes from the remainder of the candidate mixture; andvii) identifying nucleic acid ligands that photocrosslink to themolecule; b) contacting a second biological substance which does notcontain said target molecule that is associated with said disease withsaid nucleic acid ligand identified in step vii), wherein the nucleicacids with affinity to a molecule not associate with said disease isremoved; and c) amplifying the remaining nucleic acids with specificaffinity to said target molecule that is associated with said disease toyield a mixture of nucleic acids enriched for nucleic acids withrelatively higher affinity and specificity for binding to said targetmolecule that is associated with said disease state, whereby a nucleicacid ligand to a target molecule that is associated with a disease statein a biological substance is identified.
 4. A nucleic acid ligand ofclaim 3 further comprising one or more of the photoreactive groupsselected from the group consisting of 5-bromouracil, 5-iodouracil,5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil,5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine.